Hybrid Lipids Inspired by Extremophiles and Eukaryotes Afford Serum‐Stable Membranes with Low Leakage

This paper presents a new hybrid lipid that fuses the ideas of molecular tethering of lipid tails used by archaea and the integration of cholesterol groups used by eukaryotes, thereby leveraging two strategies employed by nature to increase lipid packing in membranes. Liposomes comprised of pure hybrid lipids exhibited a 5–30‐fold decrease in membrane leakage of small ions and molecules compared to liposomes that used only one strategy (lipid tethering or cholesterol incorporation) to increase membrane integrity. Molecular dynamics simulations reveal that tethering of lipid tails and integration of cholesterol both reduce the disorder in lipid tails and time‐dependent variance in area per lipid within a membrane, leading to tighter lipid packing. These hybrid lipid membranes have exceptional stability in serum, yet can support functional ion channels, can serve as a substrate for phospholipase enzymes, and can be used for liposomal delivery of molecules into living cells.Hybrid synthetic lipids with dramatically reduced leakage properties incorporate many structural features used by Nature to generate stable membranes. Covalent attachment of cholesterol groups to membrane‐spanning tetraether lipids makes it possible to generate stable liposomes with low permeability, while retaining the possibility to support functional biomolecules and deliver liposome‐encapsulated molecules to living cells.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/137415/1/chem201701378.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137415/2/chem201701378-sup-0001-misc_information.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137415/3/chem201701378_am.pd

(6R)-2-tert-Butyl-6-[(4R,5S)-3-isopropyl-4-methyl-5-phenyl­oxazolidin-2-yl]phenol

In the title compound, C23H31NO2, the lone pair on the nitro­gen atom is oriented to facilitate intra­molecular hydrogen bonding with the hydr­oxy group residing on the phenyl substituent. The five-membered ring adopts an envelope confornmation with the O atom at the flap. The absolute stereochemistry was verified by measurement of optical activity using a digital polarimeter

Design and Synthesis of Archaea-inspired Tetraether Lipids

Maintaining the correct ion homeostasis across membranes is a major challenge in both nature and artificial systems. Archaea, have evolved to solve membrane permeability problems to survive in extreme environments by incorporating unique structural features found in their lipid. Specifically, inclusion of phytanyl side chains, ether glycerol linkages, tethering of lipids, cycloalkanes, and different polar lipid headgroups into their lipid membrane are believed to contribute to membrane stability. We sought to gain a better understanding of the functional benefits attributed to these structural features to membrane stability to design a new class of synthetic Archaea inspired lipid membranes that can be used to overcome limitations (i.e. unstable in serum environment, high background leakage, and prone to hydrolysis) found in current lipid based technologies. Leakage experiments revealed liposomes made from GMGTPC (glycerol monoalkyl glycerol tetraether lipid with phosphatidylcholine headgroup) demonstrated a two order magnitude reduction in membrane leakage to small ions when compared with liposomes made from EggPC. Additionally, liposomes composed of GMGTPC-CH (cyclohexane integrated) lipid displayed an additional 40% decrease in membrane leakage to small ions when compared with liposomes made from GMGTPC lipids. Furthermore, leakage experiments revealed a higher degree of tolerance to headgroup modifications to membrane leakage for liposomes made from GMGT lipid analogs when compared with liposomes made from POPC. After designing an optimal tetraether lipid scaffold that incorporates key Archaeal structural features for membrane leakage, we explored to integrate strategies employed by eukaryotes to improve membrane properties (i.e. addition of cholesterol). Liposomes made from the hybrid lipid, GcGTPC-CH, displayed a five-fold decrease in membrane leakage when compared with liposomes made from GMGTPC-CH, while maintaining functional membrane properties similar to membranes made from diacyl lipids. Lastly, we engineered a thiol responsive hybrid lipid, GcGT(S-S)PC-CH, that demonstrated similar membrane stability in serum as GcGTPC-CH. Gratifyingly, doxorubicin loaded liposomes composed of GcGT(S-S)PC-CH liposomes displayed a 4 or 20-fold increase in toxicity to HeLa cells when compared with liposomes made from GcGTPC-CH or Doxil, respectively. This work represents a first step towards development of stimuli-responsive tetraether lipids that may offer advantages in membrane properties to be used in cancer therapy

Design and Synthesis of Archaea-inspired Tetraether Lipids

Maintaining the correct ion homeostasis across membranes is a major challenge in both nature and artificial systems. Archaea, have evolved to solve membrane permeability problems to survive in extreme environments by incorporating unique structural features found in their lipid. Specifically, inclusion of phytanyl side chains, ether glycerol linkages, tethering of lipids, cycloalkanes, and different polar lipid headgroups into their lipid membrane are believed to contribute to membrane stability. We sought to gain a better understanding of the functional benefits attributed to these structural features to membrane stability to design a new class of synthetic Archaea inspired lipid membranes that can be used to overcome limitations (i.e. unstable in serum environment, high background leakage, and prone to hydrolysis) found in current lipid based technologies. Leakage experiments revealed liposomes made from GMGTPC (glycerol monoalkyl glycerol tetraether lipid with phosphatidylcholine headgroup) demonstrated a two order magnitude reduction in membrane leakage to small ions when compared with liposomes made from EggPC. Additionally, liposomes composed of GMGTPC-CH (cyclohexane integrated) lipid displayed an additional 40% decrease in membrane leakage to small ions when compared with liposomes made from GMGTPC lipids. Furthermore, leakage experiments revealed a higher degree of tolerance to headgroup modifications to membrane leakage for liposomes made from GMGT lipid analogs when compared with liposomes made from POPC. After designing an optimal tetraether lipid scaffold that incorporates key Archaeal structural features for membrane leakage, we explored to integrate strategies employed by eukaryotes to improve membrane properties (i.e. addition of cholesterol). Liposomes made from the hybrid lipid, GcGTPC-CH, displayed a five-fold decrease in membrane leakage when compared with liposomes made from GMGTPC-CH, while maintaining functional membrane properties similar to membranes made from diacyl lipids. Lastly, we engineered a thiol responsive hybrid lipid, GcGT(S-S)PC-CH, that demonstrated similar membrane stability in serum as GcGTPC-CH. Gratifyingly, doxorubicin loaded liposomes composed of GcGT(S-S)PC-CH liposomes displayed a 4 or 20-fold increase in toxicity to HeLa cells when compared with liposomes made from GcGTPC-CH or Doxil, respectively. This work represents a first step towards development of stimuli-responsive tetraether lipids that may offer advantages in membrane properties to be used in cancer therapy

Evaluation of tetraether lipid-based liposomal carriers for encapsulation and retention of nucleoside-based drugs.

Although liposomal nanoparticles are one of the most versatile class of drug delivery systems, stable liposomal formulation of small neutral drug molecules still constitutes a challenge due to the low drug retention of current lipid membrane technologies. In this study, we evaluate the encapsulation and retention of seven nucleoside analog-based drugs in liposomes made of archaea-inspired tetraether lipids, which are known to enhance packing and membrane robustness compared to conventional bilayer-forming lipids. Liposomes comprised of the pure tetraether lipid generally showed improved retention of drugs (up to 4-fold) compared with liposomes made from a commercially available diacyl lipid. Interestingly, we did not find a significant correlation between the liposomal leakage rates of the molecules with typical parameters used to assess lipophilicity of drugs (such logD or topological polar surface area), suggesting that specific structural elements of the drug molecules can have a dominant effect on leakage from liposomes over general lipophilic character

Cyclohexane Rings Reduce Membrane Permeability to Small Ions in Archaea-Inspired Tetraether Lipids.

Extremophile archaeal organisms overcome problems of membrane permeability by producing lipids with structural elements that putatively improve membrane integrity compared to lipids from other life forms. Herein, we describe a series of lipids that mimic some key structural features of archaeal lipids, such as: 1) single tethering of lipid tails to create fully transmembrane tetraether lipids and 2) the incorporation of small rings into these tethered segments. We found that membranes formed from pure tetraether lipids leaked small ions at a rate that was about two orders of magnitude slower than common bilayer-forming lipids. Incorporation of cyclopentane rings into the tetraether lipids did not affect membrane leakage, whereas a cyclohexane ring reduced leakage by an additional 40 %. These results show that mimicking certain structural features of natural archaeal lipids results in improved membrane integrity, which may help overcome limitations of many current lipid-based technologies