Synthesis and fluorescence properties of new fluorescent, polymerizable, metal-chelating lipids

Liposomes incorporating fluorescent, metal-chelating lipids find applications in molecular recognition of peptides, 2D protein recrystallization, protein targeting, and biological sensing. It would be advantageous to combine the usefulness of polymerizable, metal-chelating lipids and fluorescent lipids. Herein, we report the synthesis and fluorescence properties of several fluorescent, polymerizable, metal-chelating lipids. They have been successfully incorporated into liposomes and then polymerized. These lipids can be used as membrane probes to study the polymerizable liposomes in the unpolymerized state and to investigate lipid redistribution during polymerization. In addition, if a luminescent metal ion (e.g., Eu(3+), Tb(3+), etc.) is used to complex the headgroup, the lipids can probe the membrane interior and exterior simultaneously.     

Polymerization of liposomes composed of diene-containing lipids by radical initiators. II. Polymerization of monodiene-type lipids as liposomes

1-Palmitoyl-2(2,4-octadecadienoyl)-sn-glycero-3-phosphocholine (POPC), a polymerizable lipid that contains one diene group in only a 2-acyl chain, was polymerized as liposome in an aqueous medium. Polymerization was initiated by water-insoluble azobisisobutyronitrile (AIBN), or water-soluble azobis(2-amidinopropane) dihydrochloride (AAPD). AIBN was mixed with monomeric lipids, and the mixture was dispersed in an aqueous medium by sonication to prepare AIBN-containing monomeric lipid liposomes. On the other hand, AAPD was simply added to the liposome suspension. The POPC liposomes were easily polymerized by the addition of AAPD, a water-soluble radical initiator, but few were polymerized by AIBN. The results suggested that the diene group in the 2-acyl chain was in an aqueous phase and, therefore, easily polymerized by a water-soluble radical initiator. The polymerized POPC liposomes were revealed to be more stable than those of monomeric ones because the scattered-light intensity from the polymerized POPC liposome suspension changed a little by the addition of Triton X-100. For only the polymerized ones, the liposome structure was confirmed by TEM after addition of an excess amount of Triton X-100.     

Langmuir-Blodgett films of fluorinated glycolipids and polymerizable lipids and their phase separating behavior

This paper describes the phase separating behavior of Langmuir monolayers from mixtures of different lipids that (i) either carry already a glycopeptide recognition site or can be easily modified to carry one and (ii) polymerizable lipids. To ensure demixing during compression, we used fluorinated lipids for the biological headgroups and hydrocarbon based lipids as polymerizable lipids. As a representative for a lipid monomer, which can be polymerized in the hydrophilic headgroup, a methacrylic monomer was used. As a monomer, which can be polymerized in the hydrophobic tail, a lipid with a diacetylene unit was used (pentacosadiynoic acid, PDA). The fluorinated lipids were on the one hand a perfluorinated lipid with three chains and on the other hand a partially fluorinated lipid with a T(N)-antigen headgroup. The macroscopic phase separation was observed by Brewster angle microscopy, whereas the phase separation on the nanoscale level was observed by atomic force microscopy. It turned out that all lipid mixtures showed (at least) a partial miscibility of the hydrocarbon compounds in the fluorinated compounds. This is positive for pattern formation, as it allows the formation of small demixed 2D patterned structures during crystallization from the homogeneous phase. For miscibility especially a liquid analogue phase proved to be advantageous. As lipid 3 with three fluorinated lipid chains (very stable monolayer) is miscible with the polymerizable lipids 1 and 2, it was mostly used for further investigations. For all three lipid mixtures, a phase separation on both the micrometer and the nanometer level was observed. The size of the crystalline domains could be controlled not only by varying the surface pressure but also by varying the molar composition of the mixtures. Furthermore, we showed that the binary mixture can be stabilized via UV polymerization. After polymerization and subsequent expansion of the barriers, the locked-in polymerized structures are stable even at low surface pressures (10 mN/m), where the unpolymerized mixture did not show any segregation.     

Synthesis of metal-chelating lipids to sensitize lanthanide ions

Sensitization of lanthanide ions is important for lanthanide ion-based assays and sensing. To the best of our knowledge, there are very few reports of lanthanide ion sensitization after it is incorporated into the liposome surface. This paper describes the syntheses of several saturated and polymerizable metal-chelating lipids based on chelidamic acid. The lipids are synthesized either from (S)-ornithine or racemic 2,3-diaminopropanoic acid. These lipids as well as polymerized liposomes incorporating these lipids sensitize lanthanide ions. Liposomes from the lipid 18-Eu(3+) provided a probe that relies not only on the emission wavelengths of Eu(3+) but also on a reproducible lifetime that can be used for protein identification.     

In vitro and in vivo stability of polymerized mixed liposomes composed of 2,4‐octadecadienoyl groups of phospholipids

The in vitro stability, under freeze–thawing procedures, and in vivo degradation, in rat spleen, of two types of polymerized liposomes were examined: 1,2‐bis‐[2E, ­4E) ‐ octadecadienoyl] ‐ sn ‐ glycero ‐ 3 ‐ phosphocholine (DODPC) and 1‐acyl‐2‐[(2E, 4E)‐octadecadienoyl]‐sn‐glycero‐3‐phosphocholine (AODPC) were used as polymerizable phospholipids. The lipid composition of the liposomes was prepared as DODPC/Chol/SA (Chol = cholesterol, SA = stearicacid), AODPC/Chol/SA (7/7/2 by molar ratio), AODPC/DPPC/Chol/SA (3.5/3.5/7/2 by molar ratio). The liposomes were extruded through a 0.2 µm polycarbonate‐ filter to obtain the approximate particle size of 0.2 µm, and then irradiated with γ‐rays. Hemoglobin‐encapsulated liposomes were also prepared in the same manner with concentrated hemoglobin (Hb) solution. The DODPC/Chol/SA liposome exhibited no trace of particle size change nor Hb leakage. Although not as excellent as the former, the AODPC‐base liposome showed slightly diameter change (below 7.5%) with a substantial abatement of Hb leakage (<3.5%). Transmission electron microscopy observation of spleens also revealed more efficient degradability with AODPC/DPPC/Chol/SA liposome than with DODPC/Chol/SA liposome. Hb‐encapsulated AODPC/DPPC/Chol/SA liposome, after five freeze–thawing cycles, attained an Hb leakage below 3.5% with a particle size change of 0.7–7.5%, and reduced the spleen retention compared with the DODPC‐base liposome. These results suggest that AODPC/DPPC/Chol/SA liposome can be used as a long‐term preservable blood substitute. Copyright © 2000 John Wiley & Sons, Ltd.     

Membrane-forming properties of pseudoglyceryl backbone based gemini lipids possessing oxyethylene spacers

Five pseudoglyceryl backbone based gemini lipids possessing varying lengths of oxyethylene [(-CH2-O-CH2-)n] spacers between cationic ammonium head groups have been synthesized, where n varies from 1 to 5. The membrane-forming properties of these gemini cationic lipids have been investigated. All the gemini lipids formed stable suspensions in water. The presence of membranous aggregates in such lipid suspensions was evidenced by transmission electron microscopy. The membrane-forming characteristics of these gemini lipids were compared with those of the corresponding monomeric lipid with one head group to understand the effect of lipid dimerization. The lipid suspensions were further characterized by dynamic light scattering and zeta potential measurements. Except for the gemini lipid with -CH2-CH2-O-CH2-CH2- spacer (2a), zeta potential of aggregates of all other gemini lipids were significantly greater than that of monomeric lipid suspensions. X-ray diffraction studies with lipid cast films revealed the increase in membrane bilayer width with increase in the length of the spacer (-CH2-O-CH2-)n. Clear thermotropic phase transitions typical of membranous assemblies were observed for all the lipid suspensions by high sensitivity differential scanning calorimetry. Aggregates of gemini lipid 2a bearing one oxyethylene [-(CH2-CH2-O-CH2-CH2)-] unit between headgroups manifested the highest phase transition temperature as compared to other gemini analogues as well as that of monomeric lipid 1. The phase transitions were reversible and exhibited large hysteresis, indicating that the observed phase transitions were of first order. To probe the surface hydration of these membranous aggregates, Paldan fluorescence studies were performed. These studies indicated the high polarity of the vesicular surface of gemini lipid 2a both in the gel and fluid melted phase as compared to vesicles of other gemini lipids.     

Liposome chromatography: liposomes immobilized in gel beads as a stationary phase for aqueous column chromatography

Liposomes have been used as a stationary phase for column chromatography with an aqueous mobile phase. They were immobilized in the pores of carrier gel beads by two methods: (A) hydrophobic ligands were coupled to the matrix of gel beads, which then were packed into a column and liposomes were applied and became associated with the ligands by hydrophobic interaction; and (B) phospholipids and detergent were dialysed in the presence of gel beads; many of the liposomes that formed in the pores of the beads were sterically immobilized by the gel matrix. Proteoliposomes containing red cell glucose transport protein in the lipid bilayers were immobilized in a column by method A. This column retained D-glucose longer than L-glucose. In contrast to L-glucose, D-glucose was transported into and out of the immobilized liposomes, causing an increased retention. Liposomes with (stearylamine)+ or (phosphatidylserine)- in their lipid bilayers were immobilized by method B and the gel beads were packed into a column. A protein of opposite charge was applied in excess. Under suitable conditions, the protein molecules became close-packed on the liposome surfaces. Ion-exchange chromatographic experiments with proteins showed that these sterically immobilized liposomes were also stable enough to be used as a stationary phase. The loss of lipids was 5-23% in the first run at high protein load and with sodium chloride gradient elution but was lower in subsequent runs. It is proposed that water-soluble molecules can be separated and their interactions with liposome surfaces studied by chromatography on immobilized liposomes in detergent-free aqueous solution. Membrane proteins can be inserted and ligands can be anchored in the lipid bilayers for chromatographic purposes.     

Syntheses, transfection efficacy and cell toxicity properties of novel cholesterol-based gemini lipids having hydroxyethyl head group

We have synthesized five new cholesterol based gemini cationic lipids possessing hydroxyethyl (-CH(2)CH(2)OH) function on each head group, which differ in the length of the polymethylene spacer chain. These gemini lipids are important for gene delivery processes as they possess pre-optimized molecular features, e.g., cholesterol backbone, ether linkage and a variable spacer chain between both the headgroups of the gemini lipids. Cationic liposomes were prepared from each of these lipids individually and as a mixture of individual cationic gemini lipid and 1,2-dioleoyl phosphatidylethanolamine (DOPE). Each gemini lipid based formulation induced better transfection activity than that of their monomeric counterpart. One such gemini lipid with a -(CH(2))(12)- spacer, HG-12, showed dramatic increase in the mean fluorescence intensity due to the expression of green-fluorescence protein (GFP) in the presence of 10% FBS compared to the conditions where there was no serum. Other gemini lipids retained their gene transfection efficiency without any marked decrease in the presence of serum. The only exception was seen with the gemini with a -(CH(2))(3)- spacer, HG-3, which on gene transfection in the presence of 10% FBS lost ~70% of its transfection efficiency. Overall the gemini lipid with a -(CH(2))(5)- spacer, HG-5, showed the highest transfection activity at N/P (lipid/DNA) ratio of 0.5 and lipid : DOPE molar ratio of 2. Upon comparison of the relevant parameters, e.g., %-transfected cells, the amount of DNA transfected to each cell and %-cell viability all together against Lipofectamine 2000, one of the best commercial transfecting agents, the optimized lipid formulation based on DOPE/HG-5 was found to be comparable. In terms of its ability to induce gene-transfer in the presence of serum and shelf-life DOPE/HG-5 liposome was found to be superior to its commercial counterpart. Confocal imaging analysis confirmed that in the presence of 10% serum using a Lipid : DOPE of 1 : 4 and N/P charge ratio of 0.75 with 1.2 μg DNA per well, HG-5 is better than Lipofectamine 2000.     

Enhanced intravenous transgene expression in mouse lung using cyclic-head cationic lipids

Herein, we report enhanced intravenous mouse lung transfection using novel cyclic-head-group analogs of usually open-head cationic transfection lipids. Design and synthesis of the new cyclic-head lipid N,N-di-n-tetradecyl-3,4-dihydroxy-pyrrolidinium chloride (lipid 1) and its higher alkyl-chain analogs (lipids 2-4) and relative in vitro and in vivo gene transfer efficacies of cyclic-head lipids 1-4 to their corresponding open-head analogs [lipid 5, namely N,N-di-n-tetradecyl-N,N-(2-hydroxyethyl)ammonium chloride and its higher alkyl-chain analogs, lipids 6-8] have been described. In stark contrast to comparable in vitro transfection efficacies of both the cyclic- and open-head lipids, lipids 1-4 with cyclic heads were found to be significantly more efficient (by 5- to 11-fold) in transfecting mouse lung than their corresponding open-head analogs (5-8) upon intravenous administration. The cyclic-head lipid 3 with di-stearyl hydrophobic tail was found to be the most promising for future applications.     

Stable polymeric nanoballoons: lyophilization and rehydration of cross-linked liposomes

The cross-linking of supramolecular assemblies of hydrated lipids is an effective method to stabilize these assemblies to disruption by surfactants or aqueous alcohol. The heterobifunctional lipids, Acryl/DenPC(16,18) and Sorb/DenPC(18,21), are examples of a new class of polymerizable lipid designed for the creation of cross-linked lipid structures. The robust nature of cross-linked liposomes was demonstrated by lyophilization of the liposomes followed by their essentially complete redispersion in water. The resulting liposomes were compared to the original sample by quasi-elastic light scattering and transmission electron microscopy. There was no major change in the size or structure of the cross-linked liposomes after rehydration of the freeze-dried powder of liposomes. Moreover, the rehydrated cross-linked liposomes continued to be resistant to surfactant solubilization. Neutral cross-linked liposomes were predominantly redispersed after freeze-drying with the aid of bath sonication. The small amount of residual liposome aggregation observed with neutral liposomes could be prevented by incorporating a surface charge into the liposome or attaching hydrophilic polymers, for example, poly(ethylene glycol), onto the liposome.     

Reconstitution of rhodopsin into polymerizable planar supported lipid bilayers: influence of dienoyl monomer structure on photoactivation

G-protein-coupled receptors (GPCRs) play key roles in cellular signal transduction and many are pharmacologically important targets for drug discovery. GPCRs can be reconstituted in planar supported lipid bilayers (PSLBs) with retention of activity, which has led to development of GPCR-based biosensors and biochips. However, PSLBs composed of natural lipids lack the high stability desired for many technological applications. One strategy is to use synthetic lipid monomers that can be polymerized to form robust bilayers. A key question is how lipid polymerization affects GPCR structure and activity. Here we have investigated the photochemical activity of bovine rhodopsin (Rho), a model GPCR, reconstituted into PSLBs composed of lipids having one or two polymerizable dienoyl moieties located in different regions of the acyl chains. Plasmon waveguide resonance spectroscopy was used to compare the degree of Rho photoactivation in fluid and poly(lipid) PSLBs. The position of the dienoyl moiety was found to have a significant effect: polymerization near the glycerol backbone significantly attenuates Rho activity whereas polymerization near the acyl chain termini does not. Differences in cross-link density near the acyl chain termini also do not affect Rho activity. In unpolymerized PSLBs, an equimolar mixture of phosphatidylethanolamine and phosphatidylcholine (PC) lipids enhances activity relative to pure PC; however after polymerization, the enhancement is eliminated which is attributed to stabilization of the membrane lamellar phase. These results should provide guidance for the design of robust lipid bilayers functionalized with transmembrane proteins for use in membrane-based biochips and biosensors.     

Hepatitis A Synthetic Peptide VP3(110-121) Miscibility with Dipalmitoylphosphatidylcholine, Dipalmitoylphosphatidylglycerol, and Stearylamine Monolayers

To prepare liposomes containing a synthetic hepatitis A virus antigen (HAV) [VP3(110-121)] as a vaccine, the miscibility of this peptide (with negative net charge) with a neutral lipid [dipalmitoylphosphatidylcholine (DPPC)], a negatively charged lipid [dipalmitoylphosphatidylglycerol (DPPG)], and a positively charged lipid [Stearylamine (SA)] was studied through compression isotherms of monolayers. Mixtures with DPPC and SA showed a low degree of interaction with the peptide, the composition of the monolayer being stable through compression. For DPPG-containing monolayers larger positive deviations from ideality were found, and the peptide was squeezed out from the monolayer at a DPPG/VP3(110-121) mole fraction of 0.8/0.2. All this suggests that besides hydrophobic interactions between the peptide and the lipid, electrostatic forces also play a role; thus it seems that neutral and positively charged lipids would be more suitable for preparing stable liposomes with VP3(110-121). Copyright 2000 Academic Press.     

Liposome formation of selectively-polymerized diene-containing phospholipids and their postpolymerization

Small and large unilamellar liposomes composed of 1,2-bis(2,4-octadecadienoyl)-sn-glycero-3-phosphorylcholine (DODPC) are prepared by sonication and extrusion, respectively. They are polymerized with water-insoluble radical initiator, azobis(isobutyronitrile) (AIBN) which can selectively polymerize diene groups in 1-acyl chains of the lipids. Polymerized liposomes are freeze-dried to obtain the polymerized liposome powder. There are two methods to redisperse lyophilized liposomes into water. The extrusion is an effective method to disperse them because the energy at extrusion is necessary only for redispersion, whereas the excess energy at sonication gives damage on liposome structure. There is no difference in stability between polymerized liposomes before and after redispersion with extrusion. DODPC polymers, obtained from free radical-initiated polymerization with AIBN, are linear and have polymerizable diene groups in 2-acyl chains. The liposome powder is therefore soluble in organic solvents. Reconstruction of polymerized liposomes is performed with lipid polymers having low or high molecular weight. The lipid polymers having high molecular weight provide stable large unilamellar liposomes by ethanol injection, but unstable small unilamellar liposomes are formed by sonication. The liposomes reconstructed from lipid polymers having low molecular weight are unstable regardless of their size. After reconstruction of liposomes selectively polymerized by AIBN, diene groups in 2-acyl chains are polymerized by water-soluble radical initiator or UV-irradiation to yield highly crosslinked structure. Their stability is improved remarkably by this postpolymerization.     

Synthesis and preliminary investigations of the siRNA delivery potential of novel, single-chain rigid cationic carotenoid lipids

The success of nucleic acid delivery requires the development of safe and efficient delivery vectors that overcome cellular barriers for effective transport. Herein we describe the synthesis of a series of novel, single-chain rigid cationic carotenoid lipids and a study of their preliminary in vitro siRNA delivery effectiveness and cellular toxicity. The efficiency of siRNA delivery by the single-chain lipid series was compared with that of known cationic lipid vectors, 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol (DC-Chol) and 1,2-dimyristoyl-sn-glyceryl-3-phosphoethanolamine (EPC) as positive controls. All cationic lipids (controls and single-chain lipids) were co-formulated into liposomes with the neutral co-lipid, 1,2-dioleolyl-sn-glycerol-3-phosphoethanolamine (DOPE). Cationic lipid-siRNA complexes of varying (+/-) molar charge ratios were formulated for delivery into HR5-CL11 cells. Of the five single-chain carotenoid lipids investigated, lipids 1, 2, 3 and 5 displayed significant knockdown efficiency with HR5-CL11 cells. In addition, lipid 1 exhibited the lowest levels of cytotoxicity with cell viability greater than 80% at all (+/-) molar charge ratios studied. This novel, single-chain rigid carotenoid-based cationic lipid represents a new class of transfection vector with excellent cell tolerance, accompanied with encouraging siRNA delivery efficiency.     

Inverse-phosphocholine lipids: a remix of a common phospholipid

Zwitterionic inverse-phosphocholine (iPC) lipids contain headgroups with an inverted charge orientation relative to phosphocholine (PC) lipids. The iPC lipid headgroup has a quaternary amine adjacent to the bilayer interface and a phosphate that extends into the aqueous phase. Neutral iPC lipids with ethylated phosphate groups (CPe) and anionic iPC lipids nonethylated phosphate groups (CP) were synthesized. The surface potential of CPe liposomes remains negative across a broad pH range and in the presence of up to 10 mM Ca(2+). CP liposomes aggregate in the presence of Ca(2+), but at a slower rate than other anionic lipids. Hydrolysis of CP lipids by alkaline phosphatases generates a cationic lipid. CPe liposomes release encapsulated anionic carboxyfluorescein (CF) 20 times faster than PC liposomes and release uncharged glucose twice as fast as PC liposomes. As such, iPC lipids afford a unique opportunity to investigate the biophysical and bioactivity-related ramifications of a charge inversion at the bilayer surface.     

Formation and regulation of fullerene-incorporation in liposomes under the phase transition temperature

The fullerene-exchange reaction from a cyclodextrin cavity to liposomes represents one of the best methods to prepare lipid membrane-incorporated [70]fullerenes (C(70)). The C(70)-exchange reaction occurred completely at temperatures above the phase transition temperature (T(m)) of the liposomes; however, lowering the temperature to below the T(m) led to C(70) aggregation outside the liposomes. This observation has limited the development of more functional LMIC(70) using a variety of liposome compositions. In this paper, this reaction was found to occur efficiently by the addition of small amounts of lipids bearing a π-moiety. The π-moieties act as a gate when hydrophobic C(70) migrates into the hydrophilic liposome surface. Therefore, the π-moieties should exist in the polar head groups of the lipids and the C(70)-exchange reaction can be controlled by pH.     

Influence of lipid composition on membrane activity of antimicrobial phenylene ethynylene oligomers

Host defense peptides (HDPs), part of the innate immune system, selectively target the membranes of bacterial cells over that of host cells. As a result, their antimicrobial properties have been under intense study. Their selectivity strongly depends on the chemical and mostly structural properties of the lipids that make up different cell membranes. The ability to synthesize HDP mimics has recently been demonstrated. To better understand how these HDP mimics interact with bilayer membranes, three homologous antimicrobial oligomers (AMOs) 1-3 with an m-phenylene ethynylene backbone and alkyl amine side chains were studied. Among them, AMO 1 is nonactive, AMO 2 is specifically active, and AMO 3 is nonspecifically active against bacteria over human red blood cells, a standard model for mammalian cells. The interactions of these three AMOs with liposomes having different lipid compositions are characterized in detail using a fluorescent dye leakage assay. AMO 2 and AMO 3 caused more leakage than AMO 1 from bacteria membrane mimic liposomes composed of PE/PG lipids. The use of E. coli lipid vesicles gave the same results. Further changes of the lipid compositions revealed that AMO 2 has selectively higher affinity toward PE/PG and E. coli lipids than PC, PC/PG or PC/PS lipids, the major components of mammalian cell membranes. In contrast, AMO 3 is devoid of this lipid selectivity and interacts with all liposomes with equal ease; AMO 1 remains inactive. These observations suggest that lipid type and structure are more important in determining membrane selectivity than lipid headgroup charges for this series of HDP mimics.     

Polymerized fluorescent liposomes incorporating lanthanide ions

This paper describes the design and synthesis of a polymerizable lipid capable of complexing lanthanide ions. The lipid has been successfully incorporated into liposomes and then polymerized. Fluorescence studies indicate that the diacetylene (unpolymerized lipid) and the conjugated alkenes (after polymerization) can be used as sensitizers for the lanthanide ion.     

Encapsulation of Hb into unsaturated lipid vesicles and γ-ray polymerization

A carbonyl hemoglobin (HbCO) solution was stirred with a mixed powder of polymerizable 1,2-bis(2,4-octadecadienoyl)-sn-glycero-3-phosphocholine (DODPC), cholesterol and stearic acid (7/7/2 by mol). The mixture was extruded through polycarbonate membrane filters (final pore size = 0.2 μm Ø). The average diameter of the resulting vesicles was 203 ± 39 nm. The [Hb]/[Lipid] ratio (the weight ratio of Hb in vesicle to lipid) increased with the Hb concentration, and decreased with the NaCl concentration. A maximum [Hb]/[lipid] ratio was observed at pH 6.9, which was the same as the isoelectric point of Hb. The vesicles were stabilized by γ-irradiation (⁶⁰Co) because the bilayer lipids bound each other to yield polyphospholipids. Denaturation of Hb by γ-irradiation was not detected. These polyphospholipid vesicles encapsulating Hb were stable even against the freeze–thaw cycles and the freeze-drying procedure.     

CHARMM‐GUI Membrane Builder toward realistic biological membrane simulations

CHARMM‐GUI Membrane Builder, http://www.charmm‐gui.org/input/membrane , is a web‐based user interface designed to interactively build all‐atom protein/membrane or membrane‐only systems for molecular dynamics simulations through an automated optimized process. In this work, we describe the new features and major improvements in Membrane Builder that allow users to robustly build realistic biological membrane systems, including (1) addition of new lipid types, such as phosphoinositides, cardiolipin (CL), sphingolipids, bacterial lipids, and ergosterol, yielding more than 180 lipid types, (2) enhanced building procedure for lipid packing around protein, (3) reliable algorithm to detect lipid tail penetration to ring structures and protein surface, (4) distance‐based algorithm for faster initial ion displacement, (5) CHARMM inputs for P₂₁ image transformation, and (6) NAMD equilibration and production inputs. The robustness of these new features is illustrated by building and simulating a membrane model of the polar and septal regions of E. coli membrane, which contains five lipid types: CL lipids with two types of acyl chains and phosphatidylethanolamine lipids with three types of acyl chains. It is our hope that CHARMM‐GUI Membrane Builder becomes a useful tool for simulation studies to better understand the structure and dynamics of proteins and lipids in realistic biological membrane environments. © 2014 Wiley Periodicals, Inc.