Tetraether archaeal lipids promote long-term survival in extreme conditions.

The sole unifying feature of the incredibly diverse Archaea is their isoprenoid-based ether-linked lipid membranes. Unique lipid membrane composition, including an abundance of membrane-spanning tetraether lipids, impart resistance to extreme conditions. Many questions remain, however, regarding the synthesis and modification of tetraether lipids and how dynamic changes to archaeal lipid membrane composition support hyperthermophily. Tetraether membranes, termed glycerol dibiphytanyl glycerol tetraethers (GDGTs), are generated by tetraether synthase (Tes) by joining the tails of two bilayer lipids known as archaeol. GDGTs are often further specialized through the addition of cyclopentane rings by GDGT ring synthase (Grs). A positive correlation between relative GDGT abundance and entry into stationary phase growth has been observed, but the physiological impact of inhibiting GDGT synthesis has not previously been reported. Here, we demonstrate that the model hyperthermophile Thermococcus kodakarensis remains viable when Tes (TK2145) or Grs (TK0167) are deleted, permitting phenotypic and lipid analyses at different temperatures. The absence of cyclopentane rings in GDGTs does not impact growth in T. kodakarensis, but an overabundance of rings due to ectopic Grs expression is highly fitness negative at supra-optimal temperatures. In contrast, deletion of Tes resulted in the loss of all GDGTs, cyclization of archaeol, and loss of viability upon transition to the stationary phase in this model archaea. These results demonstrate the critical roles of highly specialized, dynamic, isoprenoid-based lipid membranes for archaeal survival at high temperatures.

Archaeal lipids trace ecology and evolution of marine ammonia-oxidizing archaea.

Archaeal membrane lipids are widely used for paleotemperature reconstructions, yet these molecular fossils also bear rich information about ecology and evolution of marine ammonia-oxidizing archaea (AOA). Here we identified thermal and nonthermal behaviors of archaeal glycerol dialkyl glycerol tetraethers (GDGTs) by comparing the GDGT-based temperature index (TEX86) to the ratio of GDGTs with two and three cyclopentane rings (GDGT-2/GDGT-3). Thermal-dependent biosynthesis should increase TEX86 and decrease GDGT-2/GDGT-3 when the ambient temperature increases. This presumed temperature-dependent (PTD) trend is observed in GDGTs derived from cultures of thermophilic and mesophilic AOA. The distribution of GDGTs in suspended particulate matter (SPM) and sediments collected from above the pycnocline-shallow water samples-also follows the PTD trend. These similar GDGT distributions between AOA cultures and shallow water environmental samples reflect shallow ecotypes of marine AOA. While there are currently no cultures of deep AOA clades, GDGTs derived from deep water SPM and marine sediment samples exhibit nonthermal behavior deviating from the PTD trend. The presence of deep AOA increases the GDGT-2/GDGT-3 ratio and distorts the temperature-controlled correlation between GDGT-2/GDGT-3 and TEX86. We then used Gaussian mixture models to statistically characterize these diagnostic patterns of modern AOA ecology from paleo-GDGT records to infer the evolution of marine AOA from the Mid-Mesozoic to the present. Long-term GDGT-2/GDGT-3 trends suggest a suppression of today's deep water marine AOA during the Mesozoic-early Cenozoic greenhouse climates. Our analysis provides invaluable insights into the evolutionary timeline and the expansion of AOA niches associated with major oceanographic and climate changes.

Mode of carbon and energy metabolism shifts lipid composition in the thermoacidophile Acidianus.

The degree of cyclization, or ring index (RI), in archaeal glycerol dibiphytanyl glycerol tetraether (GDGT) lipids was long thought to reflect homeoviscous adaptation to temperature. However, more recent experiments show that other factors (e.g., pH, growth phase, and energy flux) can also affect membrane composition. The main objective of this study was to investigate the effect of carbon and energy metabolism on membrane cyclization. To do so, we cultivated Acidianus sp. DS80, a metabolically flexible and thermoacidophilic archaeon, on different electron donor, acceptor, and carbon source combinations (S0/Fe3+/CO2, H2/Fe3+/CO2, H2/S0/CO2, or H2/S0/glucose). We show that differences in energy and carbon metabolism can result in over a full unit of change in RI in the thermoacidophile Acidianus sp. DS80. The patterns in RI correlated with the normalized electron transfer rate between the electron donor and acceptor and did not always align with thermodynamic predictions of energy yield. In light of this, we discuss other factors that may affect the kinetics of cellular energy metabolism: electron transfer chain (ETC) efficiency, location of ETC reaction components (cytoplasmic vs. extracellular), and the physical state of electron donors and acceptors (gas vs. solid). Furthermore, the assimilation of a more reduced form of carbon during heterotrophy appears to decrease the demand for reducing equivalents during lipid biosynthesis, resulting in lower RI. Together, these results point to the fundamental role of the cellular energy state in dictating GDGT cyclization, with those cells experiencing greater energy limitation synthesizing more cyclized GDGTs.IMPORTANCESome archaea make unique membrane-spanning lipids with different numbers of five- or six-membered rings in the core structure, which modulate membrane fluidity and permeability. Changes in membrane core lipid composition reflect the fundamental adaptation strategies of archaea in response to stress, but multiple environmental and physiological factors may affect the needs for membrane fluidity and permeability. In this study, we tested how Acidianus sp. DS80 changed its core lipid composition when grown with different electron donor/acceptor pairs. We show that changes in energy and carbon metabolisms significantly affected the relative abundance of rings in the core lipids of DS80. These observations highlight the need to better constrain metabolic parameters, in addition to environmental factors, which may influence changes in membrane physiology in Archaea. Such consideration would be particularly important for studying archaeal lipids from habitats that experience frequent environmental fluctuations and/or where metabolically diverse archaea thrive.

Development of a defined medium for the heterotrophic cultivation of Metallosphaera sedula.

The heterotrophic cultivation of extremophilic archaea still heavily relies on complex media. However, complex media are associated with unknown composition, high batch-to-batch variability, potential inhibiting and interfering components, as well as regulatory challenges, hampering advancements of extremophilic archaea in genetic engineering and bioprocessing. For Metallosphaera sedula, a widely studied organism for biomining and bioremediation and a potential production host for archaeal ether lipids, efforts to find defined cultivation conditions have still been unsuccessful. This study describes the development of a novel chemically defined growth medium for M. sedula. Initial experiments with commonly used complex casein-derived media sources deciphered Casamino Acids as the most suitable foundation for further development. The imitation of the amino acid composition of Casamino Acids in basal Brock medium delivered the first chemically defined medium. We could further simplify the medium to 5 amino acids based on the respective specific substrate uptake rates. This first defined cultivation medium for M. sedula allows advanced genetic engineering and more controlled bioprocess development approaches for this highly interesting archaeon.

Vesicular and Planar Membranes of Archaea Lipids: Unusual Physical Properties and Biomedical Applications.

Liposomes and planar membranes made of archaea or archaea-like lipids exhibit many unusual physical properties compared to model membranes composed of conventional diester lipids. Here, we review several recent findings in this research area, which include (1) thermosensitive archaeosomes with the capability to drastically change the membrane surface charge, (2) MthK channel's capability to insert into tightly packed tetraether black lipid membranes and exhibit channel activity with surprisingly high calcium sensitivity, and (3) the intercalation of apolar squalane into the midplane space of diether bilayers to impede proton permeation. We also review the usage of tetraether archaeosomes as nanocarriers of therapeutics and vaccine adjuvants, as well as the biomedical applications of planar archaea lipid membranes. The discussion on archaeosomal therapeutics is focused on partially purified tetraether lipid fractions such as the polar lipid fraction E (PLFE) and glyceryl caldityl tetraether (GCTE), which are the main components of PLFE with the sugar and phosphate removed.

Certain, but Not All, Tetraether Lipids from the Thermoacidophilic Archaeon Sulfolobus acidocaldarius Can Form Black Lipid Membranes with Remarkable Stability and Exhibiting Mthk Channel Activity with Unusually High Ca2+ Sensitivity.

Bipolar tetraether lipids (BTL) have been long thought to play a critical role in allowing thermoacidophiles to thrive under extreme conditions. In the present study, we demonstrated that not all BTLs from the thermoacidophilic archaeon Sulfolobus acidocaldarius exhibit the same membrane behaviors. We found that free-standing planar membranes (i.e., black lipid membranes, BLM) made of the polar lipid fraction E (PLFE) isolated from S. acidocaldarius formed over a pinhole on a cellulose acetate partition in a dual-chamber Teflon device exhibited remarkable stability showing a virtually constant capacitance (~28 pF) for at least 11 days. PLFE contains exclusively tetraethers. The dominating hydrophobic core of PLFE lipids is glycerol dialky calditol tetraether (GDNT, ~90%), whereas glycerol dialkyl glycerol tetraether (GDGT) is a minor component (~10%). In sharp contrast, BLM made of BTL extracted from microvesicles (Sa-MVs) released from the same cells exhibited a capacitance between 36 and 39 pF lasting for only 8 h before membrane dielectric breakdown. Lipids in Sa-MVs are also exclusively tetraethers; however, the dominating lipid species in Sa-MVs is GDGT (>99%), not GDNT. The remarkable stability of BLMPLFE can be attributed to strong PLFE-PLFE and PLFE-substrate interactions. In addition, we compare voltage-dependent channel activity of calcium-gated potassium channels (MthK) in BLMPLFE to values recorded in BLMSa-MV. MthK is an ion channel isolated from a methanogenic that has been extensively characterized in diester lipid membranes and has been used as a model for calcium-gated potassium channels. We found that MthK can insert into BLMPLFE and exhibit channel activity, but not in BLMSa-MV. Additionally, the opening/closing of the MthK in BLMPLFE is detectable at calcium concentrations as low as 0.1 mM; conversely, in diester lipid membranes at such a low calcium concentration, no MthK channel activity is detectable. The differential effect of membrane stability and MthK channel activity between BLMPLFE and BLMSa-MV may be attributed to their lipid structural differences and thus their abilities to interact with the substrate and membrane protein. Since Sa-MVs that bud off from the plasma membrane are exclusively tetraether lipids but do not contain the main tetraether lipid component GDNT of the plasma membrane, domain segregation must occur in S. acidocaldarius. The implication of this study is that lipid domain formation is existent and functionally essential in all kinds of cells, but domain formation may be even more prevalent and pronounced in hyperthermophiles, as strong domain formation with distinct membrane behaviors is necessary to counteract randomization due to high growth temperatures while BTL in general make archaea cell membranes stable in high temperature and low pH environments whereas different BTL domains play different functional roles.

Total Synthesis of the Alleged Structure of Crenarchaeol Enables Structure Revision*.

Crenarchaeol is a glycerol dialkyl glycerol tetraether lipid produced exclusively in Archaea of the phylum Thaumarchaeota. This membrane-spanning lipid is undoubtedly the structurally most sophisticated of all known archaeal lipids and an iconic molecule in organic geochemistry. The 66-membered macrocycle possesses a unique chemical structure featuring 22 mostly remote stereocenters, and a cyclohexane ring connected by a single bond to a cyclopentane ring. Herein we report the first total synthesis of the proposed structure of crenarchaeol. Comparison with natural crenarchaeol allowed us to propose a revised structure of crenarchaeol, wherein one of the 22 stereocenters is inverted.

The influence of the specific growth rate on the lipid composition of Sulfolobus acidocaldarius.

Archaeal lipids are constituted of two isoprenoid chains connected via ether bonds to glycerol in the sn-2, 3 position. Due to these unique properties archaeal lipids are significantly more stable against high temperature, low pH, oxidation and enzymatic degradation than conventional lipids. Additionally, in members of the phylum Crenarchaeota condensation of two (monopolar) archaeal diether lipids to a single (bipolar) tetraether lipid as well as formation of cyclopentane rings in the isoprenoid core strongly reduce permeability of the crenarchaeal membranes. In this work we show that the Crenarchaeum Sulfolobus acidocaldarius changes its lipid composition as reaction to a shift in growth rate caused by nutrient limitation. We thereby identified a novel influencing factor for the lipid composition of S. acidocaldarius and were able to determine the effect of this factor on the lipid composition by using MALDI-MS for the semi-quantification of an archaeal lipidome: a shift in the specific growth rate during a controlled continuous cultivation of S. acidocaldarius from 0.011 to 0.035 h-1 led to a change in the ratio of diether to tetraether lipids from 1:3 to 1:5 and a decrease of the average number of cyclopentane rings from 5.1 to 4.6.

The Cell Membrane of Sulfolobus spp.-Homeoviscous Adaption and Biotechnological Applications.

The microbial cell membrane is affected by physicochemical parameters, such as temperature and pH, but also by the specific growth rate of the host organism. Homeoviscous adaption describes the process of maintaining membrane fluidity and permeability throughout these environmental changes. Archaea, and thereby, Sulfolobus spp. exhibit a unique lipid composition of ether lipids, which are altered in regard to the ratio of diether to tetraether lipids, number of cyclopentane rings and type of head groups, as a coping mechanism against environmental changes. The main biotechnological application of the membrane lipids of Sulfolobus spp. are so called archaeosomes. Archaeosomes are liposomes which are fully or partly generated from archaeal lipids and harbor the potential to be used as drug delivery systems for vaccines, proteins, peptides and nucleic acids. This review summarizes the influence of environmental parameters on the cell membrane of Sulfolobus spp. and the biotechnological applications of their membrane lipids.

Polar Lipid Fraction E from Sulfolobus acidocaldarius and Dipalmitoylphosphatidylcholine Can Form Stable yet Thermo-Sensitive Tetraether/Diester Hybrid Archaeosomes with Controlled Release Capability.

Archaeosomes have drawn increasing attention in recent years as novel nano-carriers for therapeutics. The main obstacle of using archaeosomes for therapeutics delivery has been the lack of an efficient method to trigger the release of entrapped content from the otherwise extremely stable structure. Our present study tackles this long-standing problem. We made hybrid archaeosomes composed of tetraether lipids, called the polar lipid fraction E (PLFE) isolated from the thermoacidophilic archaeon Sulfolobus acidocaldarius, and the synthetic diester lipid dipalmitoylphosphatidylcholine (DPPC). Differential polarized phase-modulation and steady-state fluorometry, confocal fluorescence microscopy, zeta potential (ZP) measurements, and biochemical assays were employed to characterize the physical properties and drug behaviors in PLFE/DPPC hybrid archaeosomes in the presence and absence of live cells. We found that PLFE lipids have an ordering effect on fluid DPPC liposomal membranes, which can slow down the release of entrapped drugs, while PLFE provides high negative charges on the outer surface of liposomes, which can increase vesicle stability against coalescence among liposomes or with cells. Furthermore, we found that the zeta potential in hybrid archaeosomes with 30 mol% PLFE and 70 mol% DPPC (designated as PLFE/DPPC(3:7) archaeosomes) undergoes an abrupt increase from -48 mV at 37 °C to -16 mV at 44 °C (termed the ZP transition), which we hypothesize results from DPPC domain melting and PLFE lipid 'flip-flop'. The anticancer drug doxorubicin (DXO) can be readily incorporated into PLFE/DPPC(3:7) archaeosomes. The rate constant of DXO release from PLFE/DPPC(3:7) archaeosomes into Tris buffer exhibited a sharp increase (~2.5 times), when the temperature was raised from 37 to 42 °C, which is believed to result from the liposomal structural changes associated with the ZP transition. This thermo-induced sharp increase in drug release was not affected by serum proteins as a similar temperature dependence of drug release kinetics was observed in human blood serum. A 15-min pre-incubation of PLFE/DPPC(3:7) archaeosomal DXO with MCF-7 breast cancer cells at 42 °C caused a significant increase in the amount of DXO entering into the nuclei and a considerable increase in the cell's cytotoxicity under the 37 °C growth temperature. Taken together, our data suggests that PLFE/DPPC(3:7) archaeosomes are stable yet potentially useful thermo-sensitive liposomes wherein the temperature range (from 37 to 42-44 °C) clinically used for mild hyperthermia treatment of tumors can be used to trigger drug release for medical interventions.

Sulfolobus acidocaldarius Microvesicles Exhibit Unusually Tight Packing Properties as Revealed by Optical Spectroscopy.

In this study, we used optical spectroscopy to characterize the physical properties of microvesicles released from the thermoacidophilic archaeon Sulfolobus acidocaldarius (Sa-MVs). The most abundant proteins in Sa-MVs are the S-layer proteins, which self-assemble on the vesicle surface forming an array of crystalline structures. Lipids in Sa-MVs are exclusively bipolar tetraethers. We found that when excited at 275 nm, intrinsic protein fluorescence of Sa-MVs at 23 °C has an emission maximum at 303 nm (or 296 nm measured at 75 °C), which is unusually low for protein samples containing multiple tryptophans and tyrosines. In the presence of 10-11 mM of the surfactant n-tetradecyl-ß-d-maltoside (TDM), Sa-MVs were disintegrated, the emission maximum of intrinsic protein fluorescence was shifted to 312 nm, and the excitation maximum was changed from 288 nm to 280.5 nm, in conjunction with a significant decrease (>2 times) in excitation band sharpness. These data suggest that most of the fluorescent amino acid residues in native Sa-MVs are in a tightly packed protein matrix and that the S-layer proteins may form J-aggregates. The membranes in Sa-MVs, as well as those of unilamellar vesicles (LUVs) made of the polar lipid fraction E (PLFE) tetraether lipids isolated from S. acidocaldarius (LUVPLFE), LUVs reconstituted from the tetraether lipids extracted from Sa-MVs (LUVMV) and LUVs made of the diester lipids, were investigated using the probe 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan). The generalized polarization (GP) values of Laurdan in tightly packed Sa-MVs, LUVMV, and LUVPLFE were found to be much lower than those obtained from less tightly packed DPPC gel state, which echoes the previous finding that the GP values from tetraether lipid membranes cannot be directly compared with the GP values from diester lipid membranes, due to differences in probe disposition. Laurdan's GP and red-edge excitation shift (REES) values in Sa-MVs and LUVMV decrease with increasing temperature monotonically with no sign for lipid phase transition. Laurdan's REES values are high (9.3-18.9 nm) in the tetraether lipid membrane systems (i.e., Sa-MVs, LUVMV and LUVPLFE) and low (0.4-5.0 nm) in diester liposomes. The high REES and low GP values suggest that Laurdan in tetraether lipid membranes, especially in the membrane of Sa-MVs, is in a very motionally restricted environment, bound water molecules and the polar moieties in the tetraether lipid headgroups strongly interact with Laurdan's excited state dipole moment, and "solvent" reorientation around Laurdan's chromophore in tetraether lipid membranes occurs very slowly compared to Laurdan's lifetime.

The Main (Glyco) Phospholipid (MPL) of Thermoplasma acidophilum.

The main phospholipid (MPL) of Thermoplasma acidophilum DSM 1728 was isolated, purified and physico-chemically characterized by differential scanning calorimetry (DSC)/differential thermal analysis (DTA) for its thermotropic behavior, alone and in mixtures with other lipids, cholesterol, hydrophobic peptides and pore-forming ionophores. Model membranes from MPL were investigated; black lipid membrane, Langmuir-Blodgett monolayer, and liposomes. Laboratory results were compared to computer simulation. MPL forms stable and resistant liposomes with highly proton-impermeable membrane and mixes at certain degree with common bilayer-forming lipids. Monomeric bacteriorhodopsin and ATP synthase from Micrococcus luteus were co-reconstituted and light-driven ATP synthesis measured. This review reports about almost four decades of research on Thermoplasma membrane and its MPL as well as transfer of this research to Thermoplasma species recently isolated from Indonesian volcanoes.

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.

Planktonic Euryarchaeota are a significant source of archaeal tetraether lipids in the ocean.

Archaea are ubiquitous in marine plankton, and fossil forms of archaeal tetraether membrane lipids in sedimentary rocks document their participation in marine biogeochemical cycles for >100 million years. Ribosomal RNA surveys have identified four major clades of planktonic archaea but, to date, tetraether lipids have been characterized in only one, the Marine Group I Thaumarchaeota. The membrane lipid composition of the other planktonic archaeal groups--all uncultured Euryarchaeota--is currently unknown. Using integrated nucleic acid and lipid analyses, we found that Marine Group II Euryarchaeota (MG-II) contributed significantly to the tetraether lipid pool in the North Pacific Subtropical Gyre at shallow to intermediate depths. Our data strongly suggested that MG-II also synthesize crenarchaeol, a tetraether lipid previously considered to be a unique biomarker for Thaumarchaeota. Metagenomic datasets spanning 5 y indicated that depth stratification of planktonic archaeal groups was a stable feature in the North Pacific Subtropical Gyre. The consistent prevalence of MG-II at depths where the bulk of exported organic matter originates, together with their ubiquitous distribution over diverse oceanic provinces, suggests that this clade is a significant source of tetraether lipids to marine sediments. Our results are relevant to archaeal lipid biomarker applications in the modern oceans and the interpretation of these compounds in the geologic record.

Ultra-high-resolution paleoenvironmental records via direct laser-based analysis of lipid biomarkers in sediment core samples.

Marine microorganisms adapt to their habitat by structural modification of their membrane lipids. This concept is the basis of numerous molecular proxies used for paleoenvironmental reconstruction. Archaeal tetraether lipids from ubiquitous marine planktonic archaea are particularly abundant, well preserved in the sedimentary record and used in several molecular proxies. We here introduce the direct, extraction-free analysis of these compounds in intact sediment core sections using laser desorption ionization (LDI) coupled to Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). LDI FTICR-MS can detect the target lipids in single submillimeter-sized spots on sediment sections, equivalent to a sample mass in the nanogram range, and could thus pave the way for biomarker-based reconstruction of past environments and ecosystems at subannual to decadal resolution. We demonstrate that ratios of selected archaeal tetraethers acquired by LDI FTICR-MS are highly correlated with values obtained by conventional liquid chromatography/MS protocols. The ratio of the major archaeal lipids, caldarchaeol and crenarchaeol, analyzed in a 6.2-cm intact section of Mediterranean sapropel S1 at 250-µm resolution (∼ 4-y temporal resolution), provides an unprecedented view of the fine-scale patchiness of sedimentary biomarker distributions and the processes involved in proxy signal formation. Temporal variations of this lipid ratio indicate a strong influence of the ∼ 200-y de Vries solar cycle on reconstructed sea surface temperatures with possible amplitudes of several degrees, and suggest signal amplification by a complex interplay of ecological and environmental factors. Laser-based biomarker analysis of geological samples has the potential to revolutionize molecular stratigraphic studies of paleoenvironments.

Concentration-Induced J-Aggregate Formation Causes a Biphasic Change in the Release of trans-Combretastatin A4 Disodium Phosphate from Archaeosomes and the Subsequent Cytotoxicity on Mammary Cancer Cells.

Combretastatin A4 disodium phosphate (CA4P) is a fluorescent, water-soluble prodrug able to induce vascular shutdown within tumors at doses less than one-tenth of the maximum tolerated dose. As a continued effort to develop efficient liposomal CA4P to treat solid tumor, we herein investigate the physical and spectroscopic properties of CA4P in aqueous solution and the mechanism of CA4P release from archaeal tetraether liposomes (archaeosomes). We found that cis-CA4P can be photoisomerized to trans-CA4P. This photoisomerization results in an increase in fluorescence intensity. Both cis- and trans-CA4P undergo fluorescence intensity self-quenching after they reach a critical concentration Cq (∼0.15-0.25 mM). Moreover, both cis- and trans-CA4P in buffer exhibit a red shift in their excitation spectrum and an increase in excitation spectrum band sharpness with increasing concentration, which can be attributed to the formation of J-aggregates. The onset of the dramatic change in excitation maximum occurs at concentrations close to Cq, suggesting that the self-quenching arises from extensive J-aggregate formation and that, when CA4P concentration exceeds Cq, J-aggregate formation begins to increase sharply. Our data also suggest that the extent of J-aggregate formation plays a critical role in CA4P release from tetraether archaeosomes and in the subsequent cytotoxicity on cultured human breast cancer MCF-7 cells. The drug leakage and cytotoxicity rate constants vary with the initial CA4P concentration entrapped inside archaeosomes in a biphasic manner, reaching a local maximum at 0.25-0.50 mM. A mechanism based on the concept of J-aggregate formation has been proposed to explain the biphasic changes in drug release and cytotoxicity with increasing drug concentration. Tetraether archaeosomes are extraordinarily stable and relatively nontoxic to animals; thus, they are promising nano drug carriers. The results obtained from this study pave the way for future development of archaeosomal CA4P to treat solid tumors.

Archaeal ether lipids improve internalization and transfection with mRNA lipid nanoparticles.

Neutral and positively charged archaeal ether lipids (AEL) have been studied for their utilization as novel delivery systems for pDNA, showing efficient immune response with a strong memory effect while lacking noticeable toxicity. Recent technological advances placed mRNA lipid nanoparticles (LNPs) at the forefront of next-generation delivery systems; however, no study has examined AELs in mRNA delivery yet. In this study, we investigated either a crude lipid extract or the purified tetraether lipid caldarchaeol from Sulfolobus acidocaldarius as potential novel excipients for mRNA LNPs. Depending on their molar share in the respective LNP, particle uptake, and mRNA expression levels could be increased by up to 10-fold in in vitro transfection experiments using both primary cell sources (HSMM) and established cell lines (Caco-2, C2C12) compared to a well-known reference formulation. This increased efficiency might be linked to a substantial effect on endosomal escape, indicating fusogenic and lyotropic features of AELs. This study shows the high value of archaeal ether lipids for mRNA delivery and provides a solid foundation for future in vivo experiments and further research.

Millennial changes and cooling trends in land surface warm-season temperatures during the Holocene.

The scarcity of proxies and calibration models for quantitatively reconstructing millennial timescale seasonal temperature tremendously constraints our understanding of the Holocene thermal variation and its driven mechanisms. Here, we established two global warm-season temperature models by applying deep learning neural network analysis to the branched tetraether membrane lipids originating from surface soil and lacustrine sediment bacteria. We utilized these optimal models in global well-dated lacustrine, peatland, and loess profiles covering the Holocene. All reconstructions of warm-season temperatures, consistent with climate model simulations, indicate cooling trends since the early Holocene, primarily induced by decreased solar radiation in the Northern Hemisphere due to the precession peak at the early. We further demonstrated that the membrane lipids can effectively enhance the future millennial seasonal temperature research, including winter temperatures, without being restricted by geographical location and sedimentary carrier.

Oral Delivery of the Vancomycin Derivative FU002 by a Surface-Modified Liposomal Nanocarrier.

Oral delivery of peptide therapeutics faces multiple challenges due to their instability in the gastrointestinal tract and low permeation capability. In this study, the aim is to develop a liposomal nanocarrier formulation to enable the oral delivery of the vancomycin-peptide derivative FU002. FU002 is a promising, resistance-breaking, antibiotic which exhibits poor oral bioavailability, limiting its potential therapeutic use. To increase its oral bioavailability, FU002 is incorporated into tetraether lipid-stabilized liposomes modified with cyclic cell-penetrating peptides on the liposomal surface. This liposomal formulation shows strong binding to Caco-2 cells without exerting cytotoxic effects in vitro. Pharmacokinetics studies in vivo in rats reveal increased oral bioavailability of liposomal FU002 when compared to the free drug. In vitro and in vivo antimicrobial activity of FU002 are preserved in the liposomal formulation. As a highlight, oral administration of liposomal FU002 results in significant therapeutic efficacy in a murine systemic infection model. Thus, the presented nanotechnological approach provides a promising strategy for enabling oral delivery of this highly active vancomycin derivative.

Highly Stable Liposomes Based on Tetraether Lipids as a Promising and Versatile Drug Delivery System.

Conventional liposomes often lack stability, limiting their applicability and usage apart from intravenous routes. Nevertheless, their advantages in drug encapsulation and physicochemical properties might be helpful in oral and pulmonary drug delivery. This study investigated the feasibility and stability of liposomes containing tetraether lipids (TEL) from Thermoplasma acidophilum. Liposomes composed of different molar ratios of TEL:Phospholipon 100H (Ph) were produced and exposed to various temperature and pH conditions. The effects on size, polydispersity index, and zeta potential were examined by dynamic and electrophoretic light scattering. Autoclaving, which was considered an additional process step after fabrication, could minimize contamination and prolong shelf life, and the stability after autoclaving was tested. Moreover, 5(6)-carboxyfluorescein leakage was measured after incubation in the presence of fetal calf serum (FCS) and lung surfactant (Alveofact). The incorporation of TEL into the liposomes significantly impacted the stability against low pH, higher temperatures, and even sterilization by autoclaving. The stability of liposomes containing TEL was confirmed by atomic force microscopy as images revealed similar sizes and morphology before and after incubation with FCS. It could be concluded that increasing the molar ratio in the TEL:Ph liposome formulations improved the structural stability against high temperature, low pH, sterilization via autoclaving, and the presence of FCS and lung surfactant.