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.

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.

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.

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.

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.

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.

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.

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.

Liposome Photosensitizer Formulations for Effective Cancer Photodynamic Therapy.

Photodynamic therapy (PDT) is a promising non-invasive strategy in the fight against that which circumvents the systemic toxic effects of chemotherapeutics. It relies on photosensitizers (PSs), which are photoactivated by light irradiation and interaction with molecular oxygen. This generates highly reactive oxygen species (such as 1O2, H2O2, O2, ·OH), which kill cancer cells by necrosis or apoptosis. Despite the promising effects of PDT in cancer treatment, it still suffers from several shortcomings, such as poor biodistribution of hydrophobic PSs, low cellular uptake, and low efficacy in treating bulky or deep tumors. Hence, various nanoplatforms have been developed to increase PDT treatment effectiveness and minimize off-target adverse effects. Liposomes showed great potential in accommodating different PSs, chemotherapeutic drugs, and other therapeutically active molecules. Here, we review the state-of-the-art in encapsulating PSs alone or combined with other chemotherapeutic drugs into liposomes for effective tumor PDT.

First Isolation and Structure Elucidation of GDNT-ß-Glu - Tetraether Lipid Fragment from Archaeal Sulfolobus Strains.

Due to their special chemical structure, tetraether lipids (TEL) represent essential elements of archaeal membranes, providing these organisms with extraordinary properties. Here we describe the characterization of a newly isolated structural element of the main lipids. The TEL fragment GDNT-ß-Glu was isolated from Sulfolobus metallicus and characterized in terms of its chemical structure by NMR- and MS-investigations. The obtained data are dissimilar to analogically derived established structures - in essence, the binding relationships in the polar head group are re-determined and verified. With this work, we provide an important contribution to the structure elucidation of intact TEL also contained in other Sulfolobus strains such as Solfulobus acidocaldarius and Sulfolobus solfataricus.

Stabilized tetraether lipids based particles guided prophyrins photodynamic therapy.

Photodynamic therapy (PDT) that involves ergonomically delivered light in the presence of archetypical photosensitizer such as Protoporphyrin IX (PpIX) is a time-honored missile strategy in cancer therapeutics. Yet, the premature release of PpIX is one of the most abundant dilemma encounters the therapeutic outcomes of PDT due to associated toxicity and redistribution to serum proteins. In this study, ultrastable tetraether lipids (TELs) based liposomes were developed. PpIX molecules were identified to reside physically in the monolayer; thereby the inherent π-π stacking that leads to aggregation of PpIX in aqueous milieu was dramatically improved. TEL29.9 mol% and TEL62mol% based liposomes revealed PpIX sustained release diffusion pattern from spherical particles as confirmed by converged fitting to Baker & Lonsdale model. Stability in presence of human serum albumins, a key element for PDT accomplishment was emphasized. The epitome candidates were selected for vascular photodynamic (vPDT) in in-Ovo chick chorioallantoic membrane. Profoundly, TEL62mol% based liposomes proved to be the most effective liposomes that demonstrated localized effect within the irradiated area without eliciting quiescent vasculatures damages. Cellular photodynamic therapy (cPDT) revealed that various radiant exposure doses of 134, 202, 403 or 672 mJ.cm-2 could deliberately modulate the photo-responses of PpIX in TEL-liposomes.

Gene deletions leading to a reduction in the number of cyclopentane rings in Sulfolobus acidocaldarius tetraether lipids.

The cell membrane of (hyper)thermophilic archaea, including the thermoacidophile Sulfolobus acidocaldarius, incorporates dibiphytanylglycerol tetraether lipids. The hydrophobic cores of such tetraether lipids can include up to eight cyclopentane rings. Presently, nothing is known of the biosynthesis of these rings. In this study, a series of S. acidocaldarius mutants deleted of genes currently annotated as encoding proteins involved in sugar/polysaccharide processing were generated and their glycolipids were considered. Whereas the glycerol-dialkyl-glycerol tetraether core of a S. acidocaldarius tetraether glycolipid considered here mostly includes four cyclopentane rings, in cells where the Saci_0421 or Saci_1201 genes had been deleted, species containing zero, two or four cyclopentane rings were observed. At the same time, in cells lacking Saci_0201, Saci_0275, Saci_1101, Saci_1249 or Saci_1706, lipids containing mostly four cyclopentane rings were detected. Although Saci_0421 and Saci_1201 are not found in proximity to other genes putatively involved in lipid biosynthesis, homologs of these sequences exist in other Archaea containing cyclopentane-containing tetraether lipids. Thus, Saci_0421 and Saci_1201 represent the first proteins described that somehow contribute to the appearance of cyclopentane rings in the core moiety of the S. acidocaldarius glycolipid considered here.

Oral delivery of vancomycin by tetraether lipid liposomes.

Despite the outstanding progress in modern medicine, the oral delivery of peptide drugs is limited until today due to their instability in the gastrointestinal tract and low mucosa penetration. To overcome these hurdles, liposomes containing the specific tetraether lipid GCTE (glycerylcaldityltetraether lipid) were examined. For this purpose, the glycopeptide antibiotic vancomycin was used as model substance and liposomes were prepared by DAC (dual assymetric centrifugation). These liposomes showed a size and polydispersity index comparable to standard liposomes. A high encapsulation efficiency of 58.53±1.76% of the peptide drug vancomycin could be obtained as detected by HPLC. FCS analysis showed that in average each liposome contains 30 molecules of vancomycin. TEM and Cryo-EM micrographs verified the size and lamellarity of the liposomal formulations. Cytotoxicity tests in Caco-2 cells showed no significant cytotoxicity for all liposomal concentrations tested, indicating the good tolerability of these formulations. Furthermore, the use of sucrose as lyoprotector enabled the long term storage of the liposomal formulation for at least three months. The potency of this drug delivery system could be proven in an animal model using Wistar rats. One hour after oral application, 4.82±0.56% of the administered dose of vancomycin could be found in the blood as detected by immunoassay measurements. This transport did also not affect the integrity of the peptide as verified by immunoassay measurements. In combination with long term storage stability, this formulation appears to be a promising delivery system for oral application of peptide drugs.

Archaeal lipids in oral delivery of therapeutic peptides.

Archaea contain membrane lipids that differ from those found in the other domains of life (Eukarya and Bacteria). These lipids consist of isoprenoid chains attached via ether bonds to the glycerol carbons at the sn-2,3 positions. Two types of ether lipids are known, polar diether lipids and bipolar tetraether lipids. The inherent chemical stability and unique membrane-spanning characteristics of tetraether lipids render them interesting for oral drug delivery purposes. Archaeal lipids form liposomes spontaneously (archaeosomes) and may be incorporated in conventional liposomes (mixed vesicles). Both types of liposomes are promising to protect their drug cargo, such as therapeutic peptides, against the acidic environment of the stomach and proteolytic degradation in the intestine. They appear to withstand lipolytic enzymes and bile salts and may thus deliver orally administered therapeutic peptides to distant sections of the intestine or to the colon, where they may be absorbed, eventually by the help of absorption enhancers. Archaeal lipids and their semisynthetic derivatives may thus serve as biological source for the next generation oral drug delivery systems. The aim of this review is to present a systematic overview over existing literature on archaea carrying diether and tetraether lipids, lipid diversity, means of lipid extraction and purification, preparation and in vitro stability studies of archaeal lipid-based liposomal drug carriers and in vivo proof-of concepts studies.

Photo-responsive tetraether lipids based vesicles for prophyrin mediated vascular targeting and direct phototherapy.

Tetraether lipids (TELs) derived from the thermoacidophilic archaeon Sulfolobus acidocaldarius are dominated by polyisoprenoid skeleton. The unique molecular stability of TELs is attributed to the presence of cyclopentane rings, methyl side groups and sugar residues that create extensive hydrogen bond network. In addition, the presence of ether linkages and the lacking of double bonds make them an epitome candidate for photodynamic therapy (PDT). A subtle blend of formulation design to trigger efficient photo responses of protoporphyrin IX (PpIX) exploiting TELs was developed. The platform has demonstrated in principle a practical potential in PDT in terms of prompt Vascular Targeting Photodynamic therapy (VTP) in-ovo chick chorioallantoic membrane (CAM) model. Short PpIX-light interval was associated with thrombosis and massive vascular occlusion in and out the irradiated area after TEL9mol% liposomes have been intravenously injected. Profoundly, TEL62mol% liposomes have proved to be the most effective liposomes that demonstrated localized suppression of angiogenesis in the irradiated area without quiescent vasculature damage. The massive thrombotic effect was no longer observed and eventually the chick has survived. After long PpIX-light interval, TEL62mol% has deliberately gained metronomic PDT at low rate of PpIX dosimetry and the radiant exposure doses in human ovarian carcinoma (SKOV-3) cells as determined by PpIXIC50. These findings could be explained by the fact that TELs impart a remarkable stability to the liposomal bilayer that makes them a potential platform for photodynamic applications.

A liposomal formulation for the oral application of the investigational hepatitis B drug Myrcludex B.

The aim of this study was the development of a liposomal formulation containing specific tetraether lipids for the oral administration of the investigational hepatitis B peptide drug Myrcludex B. For this purpose, tetraether lipids were extracted from the extremophilic archaeon Sulfolobus acidocaldarius and purified in order to obtain the desired glycerylcaldityltetraether lipids (GCTE). Myrcludex B was synthesized by solid-phase synthesis and incorporated into liposomes containing 5mol% of GCTE. These liposomes showed a size, polydispersity index and zeta potential comparable to the standard liposomes. Cryo-EM micrographs of both liposomal formulations displayed low lamellarity, the prerequisite for high drug loading capacity. Long term storage of the GCTE-liposomes was achieved by freeze-drying using 100-500mM sucrose or trehalose as lyoprotectors. The lyophilized product showed high stability with a recovery rate of 82.7±1.6% of intact Myrcludex B observed after storage for 3months at -20°C as compared to a recovery rate of 83.3±1.3% directly after the freeze-drying process. In vivo, the GCTE-liposomal formulation led to substantial enhancement of the liver uptake of iodine-131-labeled Myrcludex B in Wistar rats. 3h after oral application, approximately 7% of the initial dose (corresponding to a 3.5-fold increase compared to the free peptide) could be detected in the liver. In summary, the GCTE-liposomes enabled efficient oral administration of Myrcludex B and provided long term storage by freeze-drying.