International Journal of Pharmaceutics 493 (2015) 63–69
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Liposomes containing lipids from Sulfolobus islandicus withstand intestinal bile salts: An approach for oral drug delivery? Sara Munk Jensena,b , Camilla Jahn Christensenb,1, Julie Maria Petersenb,1, Alexander H. Treuscha , Martin Brandlb,* a b
Department of Biology and Nordic Center for Earth Evolution, University of Southern Denmark, Odense, Denmark Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 June 2015 Received in revised form 8 July 2015 Accepted 9 July 2015 Available online 17 July 2015
In an attempt to design an oral drug delivery system, suited to protect labile drug compounds like peptides and proteins against the harsh environment in the stomach and upper intestine, we have prepared liposomes from phospholipids, cholesterol and archaeal lipids. As source for the archaeal lipids we used Sulfolobus islandicus, a hyperthermophilic archaeon, whose lipids have not been used in liposomes before. Culturing conditions and extraction protocols for its membrane lipids were established and the lipid composition of the crude lipid extract was characterized. The extracted membrane lipid fraction of S. islandicus consisted primarily of diether lipids with only a small fraction of tetraether lipids. Small unilamellar liposomes with 18% (mol/mol) of crude archaeal lipid extract were from S. islandicus were produced, for the first time and proven to be stabilized against aggressive bile salts as determined by loss of entrapped marker (calcein). At 4.4 mM taurocholate (physiological taurocholate level) liposomes containing archaeal lipids retained entrapped marker better than liposomes made of egg phosphatidylcholine (PC) alone and to an extent similar to liposomes made of egg PC and cholesterol. Our findings showed that crude archaeal lipid extracts have, to a certain extent, stabilizing effects on liposomes similar to purified tetraether lipid fractions tested previously. ã 2015 Elsevier B.V. All rights reserved.
Keyword: Oral delivery system Bile salt Liposome Archaeal lipids Ether lipid Lipid structure Structure elucidation
1. Introduction Liposomes have reached the status of a clinically established drug delivery system primarily for parenteral delivery of certain cytostatic drugs. Their demonstrated potential in oral delivery of drugs, especially of therapeutic peptides and proteins, in contrast, has not been transferred into clinical use so far (Karsdal et al., 2015). Here, the function of the liposomes is to protect therapeutic agents from premature degradation in the acidic and proteolytic environment of the stomach and upper intestine. In the lower intestine and colon absorption of a range of peptides and even small proteins in therapeutically relevant amounts appears feasible (Hamman et al., 2005; Parmentier et al., 2011b). A key requirement for a successful oral delivery system is thus reasonable stability of the carrier in the gastrointestinal (GI) tract, in order to offer protection to the encapsulated peptide/protein
* Corresponding author at: Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark. E-mail address: [email protected] (M. Brandl). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijpharm.2015.07.026 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
(Sprott et al., 1996). In the light of this requirement, conventional liposome formulations are far from an ideal delivery system due their poor stability at low pH, susceptibility to attack by bile salts and lipolytic enzymes (Chia-Ming and Weiner, 1987; Rowland and Woodley, 1980). Different attempts have been undertaken to improve the liposome-based oral delivery e.g., through incorporation of membrane stabilizing additives and/or absorption enhancers, yet with variable success (Iwanaga et al., 1997; Katayama et al., 2003; Okada et al., 1995; Parmentier et al., 2010; Patel et al., 1982; Ravily et al., 1996). One class of membrane stabilizers, however, has been successfully employed by several groups: membrane lipids isolated from Archaea (Choquet et al., 1994; Li et al., 2011; Parmentier et al., 2014; Parmentier et al., 2011b). Archaea represent a domain of life with good adoptability to survive in hostile environments such as hot springs. Archaeal membrane lipids consist of the core structures dialkyl glycerol diether (DGD) and/or glycerol-dialkyl-glycerol tetraether (GDGT) wherein fully saturated phytanyl chains (20 or 40 carbons in length, respectively), are linked via ether bonds to the glycerol backbone(s) (De Rosa et al., 1986; Koga and Morii, 2007). While DGD-lipids form bilayer structures similar to conventional
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phospholipids, GDGT-lipids are membrane spanning. Archaeal lipids form vesicles (liposomes) either on their own (called archaeosomes) or in combination with phospholipids and/or other lipids (Choquet et al., 1994; Lelkes et al., 1983; Shimada et al., 2008). The variety of polar head groups may include phospho-, glycol-, phosphoglyco-, or polyol-groups very much as those found with normal ester phospholipids present in eukarya and bacteria. Archaeosomes prepared from the total polar lipids (TPL) of some archaea were relatively more stable when exposed to stress, than conventional, ester phospholipid, liposome formulations were (Choquet et al., 1994; Parmentier et al., 2011a; Patel et al., 2000; Patel and Sprott, 1999; Sprott et al., 1996). Additionally, the stability of archaeosomes was depending of the lipid source, i.e., from which archaeal organisms the lipid was extracted from (Choquet et al., 1994) as they naturally can contain different ratios of diether to tetraether. Archaeosomes from archaea with a high content of tetraether lipids yielded an improved stability compared with archaea with high amount of diether lipids. However it is unlikely that liposomes made exclusively from archaeal lipids will become a commercially relevant drug delivery system due to the demanding culturing conditions of archaea (e.g., high temperatures) and the, so far, labor-intensive extraction of their lipids. It would therefore be interesting to investigate if the stabilization effect could be obtained by mixing egg PC with ether lipids. Fricker and co-workers, have described that a semi-synthetic tetraether lipid originating from Sulfolobus acidocaldarius in combination with conventional phospholipids could protect therapeutic peptides to a similar extent as archaeosomes (Parmentier et al., 2011a). However, in order to aim for a commercially relevant drug delivery system it would be relevant if a crude lipid extract, which would be easier to obtain, could gain the same or better protective effect as observed with the semi-synthetic tetraether lipid. In this study we took the novel approach of incorporating a crude archaeal lipid extract into mixed liposomes composed of egg PC and cholesterol in order to test if they have a similar or even better protective effects than archaeosomes or mixed liposomes containing purified tetraether lipid. For this we have cultivated Sulfolobus islandicus, an archaeal species whose lipids have not been employed for liposomal drug delivery studies yet. Cultivation was performed in batch sizes large enough to secure supply for pilot-scale lipid extraction and liposome preparation. The lipid composition of the total lipid extract as determined by highresolution shotgun lipidomics indicated a major fraction of diether lipids and a minor fraction of tetraether lipids. Subsequently, unilamellar liposomes were prepared from the archaeal lipid extract in combination with phospholipids and cholesterol and tested for their marker-retaining properties upon stress, i.e., exposure to bile salts and fasted state simulated intestinal fluid (FaSSiF). The liposomes containing crude archaeal lipid extract were found to be more stable than those consisting of pure egg PC up to physiological taurocholate concentrations and equally stable as egg PC:cholesterol liposomes. 2. Materials and methods 2.1. Chemicals All solvents, buffer salts and chemicals were purchased from Sigma–Aldrich (Sigma–Aldrich Denmark ApS, Brøndby, DK), unless stated otherwise. Chloroform was purchased from VWR (VWR—Bie & Berntsen A/S, Søborg, DK). Chloroform and methanol in HPLC— grade were purchased from Rathburn (Rathburn Chemicals Ltd., Walkerburn, UK). Yeast extract, casamino acids and sodium hydroxide were purchased from Merck (Merck Life Sciences A/S, Hellerup DK). Simulated intestinal fluid (SIF) powder Original was purchased from Biorelevant (Biorelevant.com, Croydon UK).
Diether lipid standard, phosphatidylethanolamine (PE) O-20:0/ O-20:0 was purchased from Avanti (Avanti Polar Lipids Inc., Alabaster, AL, USA) and tetraether lipid standard, main polar lipid (MPL, Hex-GDGT-PG) was purchased from Matreya (Matreya LLC, State College PA, USA). Egg lecithin (E80, egg PC) was kindly provided by Lipoid (Lipoid GmbH, Ludwigshafen, DE). 2.2. Cultivation of S. islandicus S. islandicus Y.N.15.51 (Reno et al., 2009) was cultivated in 2 L fermenters containing 1.5 L Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) medium 182 (DSMZ, 2015). Temperature in the fermenters was regulated at 75 C and they were aerated by bubbling of 0.2 mm filtered air and stirring. To avoid evaporation, the exhaust air was run through a condenser that was chilled with cold water. After 4 days of cultivation, 1 L of the culture was removed and replaced with new medium for continuous cultivation. The cells were harvested by centrifugation at 5000 g and the pellet frozen and stored at 20 C. Prior to extraction the cell pellet was freeze dried overnight and stored at 20 C until extraction. 2.3. Extraction of ether lipids Two extraction methods were tested: (1) a method using sonication as cell disruption method as described in (Sturt et al., 2004); (2) a method based on a novel ball mill, dual asymmetric centrifuge (DAC) (Massing et al., 2008). The sonication approach comprised two sonication cycles of 10 min duration each at 90% duty (Bandelin electronic GmbH & Co KG, Berlin, Germany) for 100 mg cell pellet resuspended in 10 mL of 155 mM aqueous ammonium acetate solution:dichlormethane:methanol (0.8:2:1, v/v). The DAC cell-disruption method was performed as follows: the cell pellet was added to 10 mL of 155 mM ammonium acetate solution together with 10 g of glass beads (Ø 2 mm, Sigma–Aldrich, GmBH, Germany) and processed for 30 min at 2000 rpm at 4 C using a prototype dual asymmetric centrifuge (Zentrix 380r, Andreas Hettich GmbH & Co KG, Tuttlingen, Germany). The cell lysate was then transferred to separation funnels and dichloromethane (DCM), methanol (MeOH) and water were added to a final ratio of 2:1:0.8 (DCM:MeOH:H2O, v/v/v) and left until a clear phase separation appeared. Irrespective of the cell disruption method, the water-phase was washed twice with pure DCM and pooled with the collected DCM-phase. Then the collect DCM-phases were washed twice with water and finally DCM was removed using a rotary evaporator flushed with nitrogen at 37 C for a minimum of four hours. 2.4. Lipid-composition analysis of total lipid extract (TLE) by mass spectrometry analysis The TLE was analyzed by shotgun lipidomics as described (Jensen et al., 2015a). In brief, an aliquot of the TLE was diluted to approx. 100 mM (mean molar mass assumed to be 1400 g/mol) in chloroform:methanol (1:2, v/v). Prior to mass analysis it was further diluted twofold with 0.01% methylamine in methanol for runs in negative mode using LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a TriVersa NanoMate ion source (Advion Biosciences, Ithaca, USA). The settings for negative mode were ionization voltage at 0.96 kV and a gas pressure at 0.60 psi. Fourier transform mass spectrometry (FTMS) spectra were recorded in the mass range from 200 to 2000, 600 to 900 and 1200 to 2000. The target mass resolution was set to R = 100,000 (FWHM at m/z 400). The data analysis was performed using ALEX (analysis of lipid experiments) (Husen et al., 2013). As lock mass Hex-GDGT-PG 80;0 (m/z 1616.3640) was used.
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2.5. Preparation of liposomes Liposomes were prepared by conventional thin-film hydration method followed by sequential filter-extrusion (for details see Hinna et al., 2015). Three formulations were prepared: (A) pure egg lecithin (egg PC), (B) egg PC: cholesterol (ratio 80:20, mol%) and (C) egg PC:TLE:cholesterol (70:18:12, mol%) (Parmentier et al., 2011b). The assumed molar mass for the TLE was 1400 g/mol. The lipid films were hydrated using either 5 or 50 mM calcein solution in phosphate buffered saline (PBS, 10.5 mM phosphate, isotonic (285 mOsm), pH 6.5) to obtain a total lipid concentration of 100 mg/mL. Upon hydration, the liposomes were extruded by a Liposo Fast Basic hand extruder (Avestin Europe GmbH, Mannheim, Germany), first 7 times through a 400 nm pore-size membrane and then 21 times through a 200 nm pore-size membrane 19 mm diameter polycarbonate filters (Avestin Europe GmbH, Mannheim, Germany). The mean size of the liposomes was measured by dynamic light scattering using a Delsamax pro (Beckman Coulter GmbH, Krefled, DK) at 25 C in backscattering mode and in 90 measurement position. The samples were diluted to appropriate concentrations with PBS buffer. The settings were 6 measurements of 2 min sampling time each, assuming a viscosity value of 1.019 (water). 2.6. Liposome stress test Non-entrapped calcein was removed from the liposomes by size exclusion chromatography (SEC) using gravity-flow column chromatography (200 mm in length and 20 mm in diameter). 500 mL of liposome dispersion was used for each run, with PBS buffer as eluent and the fractions were collected manually. The fractions corresponding to liposomal and free calcein, respectively, were pooled and the amount of encapsulated and free calcein was determined by fluorescence measurements (Microplate spectrophotometer, FLUOstar omega, BMG Labtech GmbH, Ortenberg Germany). In order to avoid disturbances by turbidity, an aliquot of the liposome fraction was diluted with triton X-100 to make a final Triton concentration of a range from 0.5 to 3%, to disrupt the liposomes. The pooled liposome fractions were stored at 4 C and used within 48 h for the stress test. The stress test was performed by mixing an aliquot of the liposome dispersion (concentration range from 6 mM to 12 mM) with solutions of taurocholate in the concentration range 4.4– 8.9 mM, FaSSIF and PBS as a control. Samples were incubated for 90 min at 37 C, followed by fractionation by SEC. The calcein content within the free calcein and the liposomal calcein fraction was determined in order to calculate the loss of calcein (Eq. (1)).
Loss of Calcein % ¼ 100
3. Results and discussion 3.1. Extraction and lipid composition For future production it is important to be aware of batch-tobatch variability of the lipid composition from different cell pellets as well as the influence of the cell-disruption/lipid extraction method employed. In order to investigate this, a preliminary experiment analyzing the lipid composition of cell pellets from different cultivation batches was performed. Irrespective of the cell pellet and the extraction method used, up to four lead compounds (two diether lipid species and two tetraether lipid species) were identified in all lipid extracts (Fig. 1). The variability of lipid composition within parallel samples, derived from different cultivation batches, but treated by the same extraction method was considerable. This could indicate a certain extent of biological variability from cultivation to cultivation under the conditions used. However, a previous study (Jensen et al., 2015b, in review) showed less variability than observed here over a range of different growth temperatures, but most noteworthy a significant higher tetraether:diether lipid ratio. The culturing setups (flask vs.
Amount calcein in liposomes ½nmol 100 sum of calcein in liposome and free calcein ½nmol
An aliquot of the sample (not fractionated by SEC) was taken to determine recovery. The recovery was calculated with Eq. (2):
Recovery % ¼
Fig. 1. Lipid composition in relation to extraction methods dual asymmetric centrifugation (DAC) and sonication. A_DAC, B_DAC and C_DAC represent replicates from three cultures batches. Each batch was extracted by the two methods, respectively in three replicates. Values represent average SD n = 3, expect for *, where only one extraction replicate yielded one lipid species only.
(1)
fermenter) and lipid extraction methods differed between the two studies though, making a comparison difficult. Other potential factors influencing the lipid composition could be the time of
sum of calcein from liposome and free calcein ½nmol 100 amount of calcein in unfractionation sample ½nmol
(2)
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Table 1 Lipid composition in crude lipid extract. Core lipid
Lipid class
Lipid species
Relative amount (%)a
Diether lipid
P-DGD IP-DGD GDGT Hex2-GDGT-IP IP-GDGT
P-DGD 40;0 IP-DGD 40;0 GDGT 80;4 Hex2-GDGT-IP 80;4 IP-GDGT 80;1 IP-GDGT 80;2 IP-GDGT 80;3 IP-GDGT 80;4 IP-GDGT 80;5 P-GDGT 80;0 P-GDGT 80;2 P-GDGT 80;4 P-GDGT 80;5
92.3 3.94 3.0 2.25 0.3 0.17 0.2 0.07 0.1 0.10 0.5 0.37 0.2 0.13 2.2 1.16 0.1 0.06 0.1 0.05 0.2 0.05 0.7 0.19 0.1 0.04
Tetraether lipid
P-GDGT
a
The values represent average SD n = 3 of biological replicate. Three extraction replicates were made and two analytical replicates.
storage between extraction and analysis, which varied between the different samples, as well as potential inconsistencies of the analysis method. With all these factors involved, the question
the liposome formulation studies one predominant lipid species, the diether lipid with phosphorylated head group (92.3%) was identified (Table 1). One other phosphorylated diether lipid was
Table 2 Characterization of liposome formulations. Liposome formulation
Calcein concentration
Dia. (nm)a
EPC EPC:chol EPC:chol:TLE EPC:chol EPC:chol:TLE
5 mM 5 mM 5 mM 50 mM 50 mM
182.0 3.0 185.9 2.3 271.7 7.1 249.4 6.0 162.7 4.7
a
Values represent average SD n = 3 (three measurements of one typical liposome dispersion).
where the observed variability in lipid composition in this study comes from can't be finally answered. Compared to this, it seems as the extraction method only had minor influences on lipid composition. Based on these observations the DAC extraction method rather than the sonication method was chosen for further use because of its lower requirements in time and labor. Subsequently we scaled up the DAC method in order to extract 6 g of cell pellet per run and pooled the obtained lipid extracts for subsequent liposome preparation. In order to monitor the lipid composition, the pooled lipid extract was analyzed by shotgun lipidomics, with the results presented in Table 1. Within the pooled crude lipid extract used for
found, containing an inositol phosphate moiety (3.0%). The tetraether lipid species-fraction in the lipid extract amounted to 5% of the total lipid pool comprising 12 lipid species within four lipid classes. Like mentioned earlier, the lipid composition reported here differs quite substantially from our earlier results gained by small-scale extraction, where a higher amount of tetraether lipids was detected (Jensen et al., 2015a). The differences in the lipid profiles may be ascribed to the different lipid extraction methods, the cultivation conditions, inconsistencies in the analysis method or to the biological variability as described earlier. However, the high-resolution analysis of the crude lipid extract and the determination of the dominant lipid species will in the
Fig. 2. Comparison of loss of entrapped calcein from the three different liposome fomulations incubated with FaSSIF after 90 min at 37 C. Values represent average SD n = 3.
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Fig. 3. Release of entrapped calcein from the liposome formulation with ether lipids as a function of taurocholate concentration. Single values.
future help to verify how lipid composition and stability of liposomes are correlated. Obviously further investigations are needed to secure a lipid supply of more constant lipid composition. 3.2. Liposome characterization We compared three liposome formulations: pure egg PC, a mix of egg PC and cholesterol, and egg PC/cholesterol with the crude archaeal lipid extract. All three liposome formulations could be extruded without difficulties. The ether lipid liposomes showed a slightly larger diameter than the conventional liposomes (not containing ether lipids) (Table 2). However, for all three liposome formulations a reliable fractionation of free and entrapped marker could be observed. The fact that liposomes could be prepared from a mix of egg PC/cholesterol with 18% of crude lipid extract of S.
islandicus is in itself remarkable because earlier attempts had failed in this endeavor (Choquet et al., 1994). The only successful attempts to prepare mixed liposomes with lipids from the order Sulfolobales have used the purified polar lipid fraction E from S. acidocaldarius, which contains 3 tetraether compounds (Chang, 1994; Choquet et al., 1994) or a semi-synthetic tetraether lipid, GDNT with a modified head group (Parmentier et al., 2011a). 3.3. Loss of entrapped marker from liposomes upon exposure to bile salt Three selected liposome formulations, pure egg PC, egg PC with cholesterol and a formulation with ether lipids was initially incubated with FaSSiF medium (as described in Kloefer et al., 2010) in order to investigate if the liposomes formulation could survive
Fig. 4. Loss of entrapped calcein from the three liposome formulations after 90 min incubation at different taurocholate concentrations at 37 C. Entrapped calcein concentration 5 mM (A) or 50 mM (B). Values represent an average SD with n = 3, * = is a single experiment.
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intestinal conditions. Minor and comparable losses of calcein were observed for all three formulations (a loss of calcein from 6 to 12%) (Fig. 2). Thereby no significant differences were observed between the three formulations. All recovery values were in a range between 80% and 120%. The human intestinal fluid has a considerable variability in concentration of bile salts from 0.3 to 9.6 mM (for a recent review see Fuchs and Dressman, 2014). Having this range of bile salt concentrations in mind, we employed pure taurocholate up to more than double these concentrations in the stress tests. First the stability of the liposome formulation containing archaeal lipids was screened over a wide range of taurocholate concentrations. This pilot experiment showed that with increasing concentrations of taurocholate the loss of calcein from the liposomes increased from 6.1% (buffer as control) to 22.5% (4.4 mM), 92.9% (8.9 mM) to 98.9% (22 mM) (Fig. 3). Based on this result, the two other liposome formulations were also tested with concentrations of taurocholate up to 8.9 mM. In the control experiment with PBS there was no significant difference in the loss of calcein between the three formulations (Fig. 4A, p = 0.087). At taurocholate concentrations of 4.4 mM, the egg PC liposomes showed a considerable leakage of calcein, whereas from the two other liposome formulations almost no leakage occurred (Fig. 4A). At the highest taurocholate concentration tested (8.9 mM) a major loss of calcein was observed for all three liposome formulations. In the next step we raised the calcein load of the liposomes from 5 mM to 50 mM. With 50 mM calcein, the loss of calcein upon contact with taurocholate in general was higher than that from 5 mM calcein–liposomes, irrespective of liposome composition and taurocholate concentration (Fig. 4B). A reasonable explanation for this observation is a higher concentration gradient resulting in a higher diffusion potential. In addition we found that the osmolality for the two concentrations, 5 mM and 50 mM, was different (299.3 mOsm and 509.7 mOsm, respectively). Most likely a higher calcein load resulting in a higher osmolality inside the liposomes was triggering a more pronounced loss of encapsulated calcein. In conclusion, incorporation of crude archaeal lipid extract from S. islandicus in the liposome formulation had a stabilizing effect up to a taurocholate concentration of 4.4 mM and in FaSSIF medium as compared with liposomes made of egg PC. The stabilization effect was similar for the TLE liposomes and the egg PC:cholesterol liposomes. The crude lipid extract used for this experiment contained primarily diether lipids, suggesting that diether lipids from S. islandicus have a stabilizing effect. Previously it has been shown that archaeosomes made of 100% crude lipid extract from Thermoplasma acidophilum released in a comparable setting, i.e., upon 90 min incubation with 10 mM bile salt, almost all of the entrapped marker (Patel et al., 2000). In comparison, our mixed liposomes containing 18% crude lipid extract from S. islandicus showed similar stability against bile salts, however the exact lipid composition of the crude extract from T. acidophilum is unknown. Taken into account that it is very costly and labor-intensive to obtain and purify ether lipids, this knowledge is a step in the right direction in order to have an optimal and stable liposome formulation. However Fricker and colleagues have described that an egg PC-liposome formulation containing 9 mol% of a pure semisynthetic tetraether lipid (from S. acidocaldarius) had a stabilizing effect even at 10 mM taurocholate (>60% retained marker) at 90 min. This effect is superior to the stabilization we have observed, indicating that a single pure tetraether lipid exerts a stronger stabilizing effect to liposomes than the crude lipid extract investigated here. To employ a pure, semisynthetic tetraether lipid, however is costly and thus not suited for commercial production. In a previous study we have shown that the lipid composition of S. islandicus varies with culture conditions, especially a higher content of cyclopentane-moieties was observed with higher
culturing temperatures (Jensen et al., 2015b, in review). It could thus be investigated if crude lipid extracts obtained from S. islandicus grown under different culturing conditions would vary in their liposome stabilizing potential. It is also worth mentioning that we had observed higher percentages of tetraether lipids within crude lipid extracts from S. islandicus when we were extracting the cell pellet with a small-scale extraction method, which is suited for analytical purposes only. This result is more in line with the literature, where the main membrane lipids of S. islandicus are thought to be tetraether lipids (Jensen et al., 2015a). Set in perspective to the observation by Patel et al. (2000) that crude archaeal lipid extracts with predominantly tetraether lipids had a more pronounced stabilizing effect than those with predominantly diether lipids, there may be room for improvement with the crude lipid extract from S. islandicus. This gives rise to the hope that optimized culturing conditions as well as lipid extraction protocols can result in higher percentages of tetraether lipids at larger scale and thereby improve the stability of liposomes against bile salts. Finally, it should be mentioned, that there are several threats in the GI-tract, which the liposome formulation must resist. In this study we have focused on one threat only, the bile salts, since they have earlier been identified as the most important stressors for archaeosomes, where most of the entrapped marker was lost, in comparison to low pH and lipase activity (Patel et al., 2000). But since our ether liposome formulation only contains a smaller percentage of ether lipids it not known if the same will apply for this liposome formulation. Therefore it is relevant for future experiments to change the experimental settings by exposing the liposome formulation to more complex media containing both bile salts and lipases in order to simulate the intestinal conditions more appropriately. 4. Conclusion The current proof-of-concept study demonstrated that S. islandicus represents a potential source for biotechnological production of ether lipids. The total lipid extracts typically comprised both, diether- and tetraether-lipids in varying composition, as revealed by shotgun lipidomics. Egg PC-liposomes containing 18 mol% of the total lipid extract from S. islandicus were found to withstand in vitro exposure to bile salt (taurocholate) up to concentrations of 4 mM without substantial loss of the entrapped hydrophilic marker calcein. In contrast, conventional (egg PC) liposomes released almost their entire entrapped marker at this bile salts concentration, indicating that ether lipids from S. islandicus exert a liposome-stabilizing effect on liposomal drug carriers when it comes to bile salt-related stress. Future work will need to focus on increasing the tetraether lipid content in the crude lipid extract, studying if the recently described influence of culture conditions on ether lipid-composition and/or an optimized extraction process would yield even better membrane stabilizing effects. Finally, it remains to be investigated if these liposomestabilizing effects can be transferred into successful oral peptide delivery in vivo. Acknowledgements We thank Rachel Whitaker (University of Illinois) and Arnulf Kletzin (Technische Universität Darmstadt) for providing the Sulfolobus islandicus. We are grateful to Christer S. Ejsing, Renialdo Almeida, Albert Casanovas and Martin Hermansson for the scientific disscussion, data intepreation and help with mass spectrometry analysis. We thank Trine Johansson, Dina Skov and Tina Christiansen for technical assistance with cultivation and lipid extraction. We thank Askell Hinna for helpful discussions
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regarding data interpretation. We are grateful to Ole N. Jensen for kindly providing access to the TriVersa NanoMate. This work was supported by Danish National Research Foundation grant (DNRF53, AHT). References Chang, E.L., 1994. Unusual thermal stability of liposomes made from bipolar tetraether lipids. Biochem. Biophys. Res. Commun. 202, 673–679. Chia-Ming, C., Weiner, N., 1987. Gastrointestinal uptake of liposomes. I. In vitro and in situ studies. Int. J. Pharm. 37, 75–85. Choquet, C.G., Patel, G.B., Beveridge, T.J., Sprott, G.D., 1994. Stability of pressureextrudes liposomes made from archaeobacterial ether lipids. Appl. Microbiol. Biotechnol. 42, 375–384. De Rosa, M., Gambacorta, A., Gliozzi, A., 1986. Structure, Biosynthesis and physicochemical properties of archaebacterial lipids. Microbiol. Rev. 50, 10. DSMZ, 2015. DSMZ medie 182. Fuchs, A., Dressman, J.B., 2014. Composition and physicochemical properties of fasted-state human duodenal and jejunal fluid: a critical evaluation of the available data. J. Pharm. Sci. 103, 3398–3411. Hamman, J.H., Enslin, G.M., Kotzé, A.F., 2005. Oral delivery of peptide drugs: barriers and developments. BioDrugs 19, 165–177. Hinna, A., Steiniger, F., Hupfeld, S., Stein, P., Kuntsche, J., Brandl, M., 2015. Filterextruded liposomes revisited: a study into size distributions and morphologies in relation to lipid-composition and process parameters. J. Liposome Res. 31, 1– 10. Husen, P., Tarasov, K., Katafiasz, M., Sokol, E., Vogt, J., Baumgart, J., Nitsch, R., Ekroos, K., Ejsing, C.S., 2013. Analysis of lipid experiments (ALEX): a software framework for analysis of high-resolution shotgun lipidomics data. PloS ONE 8, e79736. Iwanaga, K., Ono, S., Narioka, K., Morimoto, K., Kakemi, M., Yamashita, S., Nango, M., Oku, N., 1997. Oral delivery of insulin by using surface coating liposomes: improvement of stability of insulin in GI tract. Int. J. Pharm. 157, 73–80. Jensen, S.M., Brandl, M., Treusch, A.H., Ejsing, C.S., 2015a. Structural characterization of ether lipids from the archaeon Sulfolobus islandicus by high-resolution shotgun lipidomics. J. Mass Spectrom. 50, 476–487. Jensen, S.M., Neesgaard, V.L., Skjoldbjerg, S.L.N., Brandl, M., Ejsing, C.S., Treusch, A. H., 2015. The effects of temperature and growth phase on the lipidomes of Sulfolobus islandicus and Sulfolobus tokodaii (in review). Karsdal, M.A., Riis, B.J., Mehta, N., Stern, W., Arbit, E., Christiansen, C., Henriksen, K., 2015. Lessons learned from the clinical development of oral peptides. Br. J. Clin. Pharmacol. 79, 720–732. Katayama, K., Kato, Y., Onishi, H., Nagai, T., Machida, Y., 2003. Double liposomes: Hypoglycemic effects of liposomal insulin on normal rats. Drug Dev. Ind. Pharm. 29, 725–731. Kloefer, B., van Hoogevest, P., Moloney, R., Kuentz, M., Leigh, M.L.S., Dressman, J., 2010. Study of a standardized taurocholate–lecithin powder for preparing the biorelevant media FeSSIF and FaSSIF. Dissolut. Technol. 17, 6–13. Koga, Y., Morii, H., 2007. Biosynthesis of ether-type polar lipids in archaea and evolutionary considerations. Microbiol. Mol. Biol. Rev. 71, 97–120.
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Lelkes, P.I., Goldenberg, D., Gliozzi, A., De Rosa, M., Gambacorta, A., Miller, I.R., 1983. Vesicles from mixtures of bipolar archaebacterial lipids with egg phosphatidylcholine. BBA Biomembr. 732, 714–718. Li, Z., Zhang, L., Sun, W., Ding, Q., Hou, Y., Xu, Y., 2011. Archaeosomes with encapsulated antigens for oral vaccine delivery. Vaccine 29, 5260–5266. Massing, U., Cicko, S., Ziroli, V., 2008. Dual asymmetric centrifugation (DAC)—a new technique for liposome preparation. J. Control. Release 125, 16–24. Okada, J., Cohen, S., Langer, R., 1995. In vitro evaluation of polymerized liposomes as an oral drug delivery system. Pharm. Res. 12, 576–582. Parmentier, J., Becker, M.M., Heintz, U., Fricker, G., 2011a. Stability of liposomes containing bio-enhancers and tetraether lipids in simulated gastro-intestinal fluids. Int. J. Pharm. 405, 210–217. Parmentier, J., Hartmann, F.J., Fricker, G., 2010. In vitro evaluation of liposomes containing bio-enhancers for the oral delivery of macromolecules. Eur. J. Pharm. Biopharm. 76, 394–403. Parmentier, J., Hofhaus, G., Thomas, S., Cuesta, L.C., Gropp, F., Schroder, R., Hartmann, K., Fricker, G., 2014. Improved oral bioavailability of human growth hormone by a combination of liposomes containing bio-enhancers and tetraether lipids and omeprazole. J. Pharm. Sci. 103, 3985–3993. Parmentier, J., Thewes, B., Gropp, F., Fricker, G., 2011b. Oral peptide delivery by tetraether lipid liposomes. Int. J. Pharm. 415, 150–157. Patel, G.B., Agnew, B.J., Deschatelets, L., Fleming, L.P., Sprott, G.D., 2000. In vitro assessment of archaeosome stability for developing oral delivery systems. Int. J. Pharm. 194, 39–49. Patel, G.B., Sprott, G.D., 1999. Archaeobacterial ether lipid liposomes (archaeosomes) as novel vaccine and drug delivery systems. Crit. Rev. Biotechnol. 19, 317–357. Patel, H.M., Stevenson, R.W., Parsons, J.A., Ryman, B.E., 1982. Use of liposomes to aid intestinal absorption of entrapped insulin in normal and diabetic dogs. BBA Gen. Subj. 716, 188–193. Ravily, V., Santaella, C., Vierling, P., 1996. Membrane permeability and stability in buffer and in human serum of fluorinated di-O-alkylglycerophosphocholinebased liposomes. Biochim. Biophys. Acta (BBA) 1285, 79–90. Reno, M.L., Held, N.L., Fields, C.J., Burke, P.V., Whitaker, R.J., 2009. Biogeography of the Sulfolobus islandicus pan-genome. Proc. Natl. Acad. Sci. U. S. A. 106, 8605– 8610. Rowland, R.N., Woodley, J.F., 1980. The stability of liposomes in vitro to pH, bile salts and pancreatic lipase. Biochim. Biophys. Acta (BBA) 620, 400–409. Shimada, H., Nemoto, N., Shida, Y., Oshima, T., Yamagishi, A., 2008. Effects of pH and temperature on the composition of polar lipids in thermoplasma acidophilum HO-62. J. Bacteriol. 190, 5404–5411. Sprott, G.D., Dicaire, C.J., Fleming, L.P., Patel, G.B., Kates, M., Foldvari, M., Langworthy, T.A., Dash, A.K., 1996. Stability of liposomes prepared from archaeobacterial lipids and phosphatidylcholine, mixtures. Cells Mater. 6, 143–155. Sturt, H.F., Summons, R.E., Smith, K., Elvert, M., Hinrichs, K.U., 2004. Intact polar membrane lipids in prokaryotes and sediments deciphered by highperformance liquid chromatography/electrospray ionization multistage mass spectrometry—new biomarkers for biogeochemistry and microbial ecology. Rapid Commun. Mass Spectrom. 18, 617–628.
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Liposomes containing lipids from Sulfolobus islandicus withstand intestinal bile salts: An approach for oral drug delivery? Sara Munk Jensena,b , Camilla Jahn Christensenb,1, Julie Maria Petersenb,1, Alexander H. Treuscha , Martin Brandlb,* a b
Department of Biology and Nordic Center for Earth Evolution, University of Southern Denmark, Odense, Denmark Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Odense, Denmark
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 June 2015 Received in revised form 8 July 2015 Accepted 9 July 2015 Available online 17 July 2015
In an attempt to design an oral drug delivery system, suited to protect labile drug compounds like peptides and proteins against the harsh environment in the stomach and upper intestine, we have prepared liposomes from phospholipids, cholesterol and archaeal lipids. As source for the archaeal lipids we used Sulfolobus islandicus, a hyperthermophilic archaeon, whose lipids have not been used in liposomes before. Culturing conditions and extraction protocols for its membrane lipids were established and the lipid composition of the crude lipid extract was characterized. The extracted membrane lipid fraction of S. islandicus consisted primarily of diether lipids with only a small fraction of tetraether lipids. Small unilamellar liposomes with 18% (mol/mol) of crude archaeal lipid extract were from S. islandicus were produced, for the first time and proven to be stabilized against aggressive bile salts as determined by loss of entrapped marker (calcein). At 4.4 mM taurocholate (physiological taurocholate level) liposomes containing archaeal lipids retained entrapped marker better than liposomes made of egg phosphatidylcholine (PC) alone and to an extent similar to liposomes made of egg PC and cholesterol. Our findings showed that crude archaeal lipid extracts have, to a certain extent, stabilizing effects on liposomes similar to purified tetraether lipid fractions tested previously. ã 2015 Elsevier B.V. All rights reserved.
Keyword: Oral delivery system Bile salt Liposome Archaeal lipids Ether lipid Lipid structure Structure elucidation
1. Introduction Liposomes have reached the status of a clinically established drug delivery system primarily for parenteral delivery of certain cytostatic drugs. Their demonstrated potential in oral delivery of drugs, especially of therapeutic peptides and proteins, in contrast, has not been transferred into clinical use so far (Karsdal et al., 2015). Here, the function of the liposomes is to protect therapeutic agents from premature degradation in the acidic and proteolytic environment of the stomach and upper intestine. In the lower intestine and colon absorption of a range of peptides and even small proteins in therapeutically relevant amounts appears feasible (Hamman et al., 2005; Parmentier et al., 2011b). A key requirement for a successful oral delivery system is thus reasonable stability of the carrier in the gastrointestinal (GI) tract, in order to offer protection to the encapsulated peptide/protein
* Corresponding author at: Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, DK-5230 Odense, Denmark. E-mail address: [email protected] (M. Brandl). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijpharm.2015.07.026 0378-5173/ ã 2015 Elsevier B.V. All rights reserved.
(Sprott et al., 1996). In the light of this requirement, conventional liposome formulations are far from an ideal delivery system due their poor stability at low pH, susceptibility to attack by bile salts and lipolytic enzymes (Chia-Ming and Weiner, 1987; Rowland and Woodley, 1980). Different attempts have been undertaken to improve the liposome-based oral delivery e.g., through incorporation of membrane stabilizing additives and/or absorption enhancers, yet with variable success (Iwanaga et al., 1997; Katayama et al., 2003; Okada et al., 1995; Parmentier et al., 2010; Patel et al., 1982; Ravily et al., 1996). One class of membrane stabilizers, however, has been successfully employed by several groups: membrane lipids isolated from Archaea (Choquet et al., 1994; Li et al., 2011; Parmentier et al., 2014; Parmentier et al., 2011b). Archaea represent a domain of life with good adoptability to survive in hostile environments such as hot springs. Archaeal membrane lipids consist of the core structures dialkyl glycerol diether (DGD) and/or glycerol-dialkyl-glycerol tetraether (GDGT) wherein fully saturated phytanyl chains (20 or 40 carbons in length, respectively), are linked via ether bonds to the glycerol backbone(s) (De Rosa et al., 1986; Koga and Morii, 2007). While DGD-lipids form bilayer structures similar to conventional
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phospholipids, GDGT-lipids are membrane spanning. Archaeal lipids form vesicles (liposomes) either on their own (called archaeosomes) or in combination with phospholipids and/or other lipids (Choquet et al., 1994; Lelkes et al., 1983; Shimada et al., 2008). The variety of polar head groups may include phospho-, glycol-, phosphoglyco-, or polyol-groups very much as those found with normal ester phospholipids present in eukarya and bacteria. Archaeosomes prepared from the total polar lipids (TPL) of some archaea were relatively more stable when exposed to stress, than conventional, ester phospholipid, liposome formulations were (Choquet et al., 1994; Parmentier et al., 2011a; Patel et al., 2000; Patel and Sprott, 1999; Sprott et al., 1996). Additionally, the stability of archaeosomes was depending of the lipid source, i.e., from which archaeal organisms the lipid was extracted from (Choquet et al., 1994) as they naturally can contain different ratios of diether to tetraether. Archaeosomes from archaea with a high content of tetraether lipids yielded an improved stability compared with archaea with high amount of diether lipids. However it is unlikely that liposomes made exclusively from archaeal lipids will become a commercially relevant drug delivery system due to the demanding culturing conditions of archaea (e.g., high temperatures) and the, so far, labor-intensive extraction of their lipids. It would therefore be interesting to investigate if the stabilization effect could be obtained by mixing egg PC with ether lipids. Fricker and co-workers, have described that a semi-synthetic tetraether lipid originating from Sulfolobus acidocaldarius in combination with conventional phospholipids could protect therapeutic peptides to a similar extent as archaeosomes (Parmentier et al., 2011a). However, in order to aim for a commercially relevant drug delivery system it would be relevant if a crude lipid extract, which would be easier to obtain, could gain the same or better protective effect as observed with the semi-synthetic tetraether lipid. In this study we took the novel approach of incorporating a crude archaeal lipid extract into mixed liposomes composed of egg PC and cholesterol in order to test if they have a similar or even better protective effects than archaeosomes or mixed liposomes containing purified tetraether lipid. For this we have cultivated Sulfolobus islandicus, an archaeal species whose lipids have not been employed for liposomal drug delivery studies yet. Cultivation was performed in batch sizes large enough to secure supply for pilot-scale lipid extraction and liposome preparation. The lipid composition of the total lipid extract as determined by highresolution shotgun lipidomics indicated a major fraction of diether lipids and a minor fraction of tetraether lipids. Subsequently, unilamellar liposomes were prepared from the archaeal lipid extract in combination with phospholipids and cholesterol and tested for their marker-retaining properties upon stress, i.e., exposure to bile salts and fasted state simulated intestinal fluid (FaSSiF). The liposomes containing crude archaeal lipid extract were found to be more stable than those consisting of pure egg PC up to physiological taurocholate concentrations and equally stable as egg PC:cholesterol liposomes. 2. Materials and methods 2.1. Chemicals All solvents, buffer salts and chemicals were purchased from Sigma–Aldrich (Sigma–Aldrich Denmark ApS, Brøndby, DK), unless stated otherwise. Chloroform was purchased from VWR (VWR—Bie & Berntsen A/S, Søborg, DK). Chloroform and methanol in HPLC— grade were purchased from Rathburn (Rathburn Chemicals Ltd., Walkerburn, UK). Yeast extract, casamino acids and sodium hydroxide were purchased from Merck (Merck Life Sciences A/S, Hellerup DK). Simulated intestinal fluid (SIF) powder Original was purchased from Biorelevant (Biorelevant.com, Croydon UK).
Diether lipid standard, phosphatidylethanolamine (PE) O-20:0/ O-20:0 was purchased from Avanti (Avanti Polar Lipids Inc., Alabaster, AL, USA) and tetraether lipid standard, main polar lipid (MPL, Hex-GDGT-PG) was purchased from Matreya (Matreya LLC, State College PA, USA). Egg lecithin (E80, egg PC) was kindly provided by Lipoid (Lipoid GmbH, Ludwigshafen, DE). 2.2. Cultivation of S. islandicus S. islandicus Y.N.15.51 (Reno et al., 2009) was cultivated in 2 L fermenters containing 1.5 L Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) medium 182 (DSMZ, 2015). Temperature in the fermenters was regulated at 75 C and they were aerated by bubbling of 0.2 mm filtered air and stirring. To avoid evaporation, the exhaust air was run through a condenser that was chilled with cold water. After 4 days of cultivation, 1 L of the culture was removed and replaced with new medium for continuous cultivation. The cells were harvested by centrifugation at 5000 g and the pellet frozen and stored at 20 C. Prior to extraction the cell pellet was freeze dried overnight and stored at 20 C until extraction. 2.3. Extraction of ether lipids Two extraction methods were tested: (1) a method using sonication as cell disruption method as described in (Sturt et al., 2004); (2) a method based on a novel ball mill, dual asymmetric centrifuge (DAC) (Massing et al., 2008). The sonication approach comprised two sonication cycles of 10 min duration each at 90% duty (Bandelin electronic GmbH & Co KG, Berlin, Germany) for 100 mg cell pellet resuspended in 10 mL of 155 mM aqueous ammonium acetate solution:dichlormethane:methanol (0.8:2:1, v/v). The DAC cell-disruption method was performed as follows: the cell pellet was added to 10 mL of 155 mM ammonium acetate solution together with 10 g of glass beads (Ø 2 mm, Sigma–Aldrich, GmBH, Germany) and processed for 30 min at 2000 rpm at 4 C using a prototype dual asymmetric centrifuge (Zentrix 380r, Andreas Hettich GmbH & Co KG, Tuttlingen, Germany). The cell lysate was then transferred to separation funnels and dichloromethane (DCM), methanol (MeOH) and water were added to a final ratio of 2:1:0.8 (DCM:MeOH:H2O, v/v/v) and left until a clear phase separation appeared. Irrespective of the cell disruption method, the water-phase was washed twice with pure DCM and pooled with the collected DCM-phase. Then the collect DCM-phases were washed twice with water and finally DCM was removed using a rotary evaporator flushed with nitrogen at 37 C for a minimum of four hours. 2.4. Lipid-composition analysis of total lipid extract (TLE) by mass spectrometry analysis The TLE was analyzed by shotgun lipidomics as described (Jensen et al., 2015a). In brief, an aliquot of the TLE was diluted to approx. 100 mM (mean molar mass assumed to be 1400 g/mol) in chloroform:methanol (1:2, v/v). Prior to mass analysis it was further diluted twofold with 0.01% methylamine in methanol for runs in negative mode using LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with a TriVersa NanoMate ion source (Advion Biosciences, Ithaca, USA). The settings for negative mode were ionization voltage at 0.96 kV and a gas pressure at 0.60 psi. Fourier transform mass spectrometry (FTMS) spectra were recorded in the mass range from 200 to 2000, 600 to 900 and 1200 to 2000. The target mass resolution was set to R = 100,000 (FWHM at m/z 400). The data analysis was performed using ALEX (analysis of lipid experiments) (Husen et al., 2013). As lock mass Hex-GDGT-PG 80;0 (m/z 1616.3640) was used.
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2.5. Preparation of liposomes Liposomes were prepared by conventional thin-film hydration method followed by sequential filter-extrusion (for details see Hinna et al., 2015). Three formulations were prepared: (A) pure egg lecithin (egg PC), (B) egg PC: cholesterol (ratio 80:20, mol%) and (C) egg PC:TLE:cholesterol (70:18:12, mol%) (Parmentier et al., 2011b). The assumed molar mass for the TLE was 1400 g/mol. The lipid films were hydrated using either 5 or 50 mM calcein solution in phosphate buffered saline (PBS, 10.5 mM phosphate, isotonic (285 mOsm), pH 6.5) to obtain a total lipid concentration of 100 mg/mL. Upon hydration, the liposomes were extruded by a Liposo Fast Basic hand extruder (Avestin Europe GmbH, Mannheim, Germany), first 7 times through a 400 nm pore-size membrane and then 21 times through a 200 nm pore-size membrane 19 mm diameter polycarbonate filters (Avestin Europe GmbH, Mannheim, Germany). The mean size of the liposomes was measured by dynamic light scattering using a Delsamax pro (Beckman Coulter GmbH, Krefled, DK) at 25 C in backscattering mode and in 90 measurement position. The samples were diluted to appropriate concentrations with PBS buffer. The settings were 6 measurements of 2 min sampling time each, assuming a viscosity value of 1.019 (water). 2.6. Liposome stress test Non-entrapped calcein was removed from the liposomes by size exclusion chromatography (SEC) using gravity-flow column chromatography (200 mm in length and 20 mm in diameter). 500 mL of liposome dispersion was used for each run, with PBS buffer as eluent and the fractions were collected manually. The fractions corresponding to liposomal and free calcein, respectively, were pooled and the amount of encapsulated and free calcein was determined by fluorescence measurements (Microplate spectrophotometer, FLUOstar omega, BMG Labtech GmbH, Ortenberg Germany). In order to avoid disturbances by turbidity, an aliquot of the liposome fraction was diluted with triton X-100 to make a final Triton concentration of a range from 0.5 to 3%, to disrupt the liposomes. The pooled liposome fractions were stored at 4 C and used within 48 h for the stress test. The stress test was performed by mixing an aliquot of the liposome dispersion (concentration range from 6 mM to 12 mM) with solutions of taurocholate in the concentration range 4.4– 8.9 mM, FaSSIF and PBS as a control. Samples were incubated for 90 min at 37 C, followed by fractionation by SEC. The calcein content within the free calcein and the liposomal calcein fraction was determined in order to calculate the loss of calcein (Eq. (1)).
Loss of Calcein % ¼ 100
3. Results and discussion 3.1. Extraction and lipid composition For future production it is important to be aware of batch-tobatch variability of the lipid composition from different cell pellets as well as the influence of the cell-disruption/lipid extraction method employed. In order to investigate this, a preliminary experiment analyzing the lipid composition of cell pellets from different cultivation batches was performed. Irrespective of the cell pellet and the extraction method used, up to four lead compounds (two diether lipid species and two tetraether lipid species) were identified in all lipid extracts (Fig. 1). The variability of lipid composition within parallel samples, derived from different cultivation batches, but treated by the same extraction method was considerable. This could indicate a certain extent of biological variability from cultivation to cultivation under the conditions used. However, a previous study (Jensen et al., 2015b, in review) showed less variability than observed here over a range of different growth temperatures, but most noteworthy a significant higher tetraether:diether lipid ratio. The culturing setups (flask vs.
Amount calcein in liposomes ½nmol 100 sum of calcein in liposome and free calcein ½nmol
An aliquot of the sample (not fractionated by SEC) was taken to determine recovery. The recovery was calculated with Eq. (2):
Recovery % ¼
Fig. 1. Lipid composition in relation to extraction methods dual asymmetric centrifugation (DAC) and sonication. A_DAC, B_DAC and C_DAC represent replicates from three cultures batches. Each batch was extracted by the two methods, respectively in three replicates. Values represent average SD n = 3, expect for *, where only one extraction replicate yielded one lipid species only.
(1)
fermenter) and lipid extraction methods differed between the two studies though, making a comparison difficult. Other potential factors influencing the lipid composition could be the time of
sum of calcein from liposome and free calcein ½nmol 100 amount of calcein in unfractionation sample ½nmol
(2)
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Table 1 Lipid composition in crude lipid extract. Core lipid
Lipid class
Lipid species
Relative amount (%)a
Diether lipid
P-DGD IP-DGD GDGT Hex2-GDGT-IP IP-GDGT
P-DGD 40;0 IP-DGD 40;0 GDGT 80;4 Hex2-GDGT-IP 80;4 IP-GDGT 80;1 IP-GDGT 80;2 IP-GDGT 80;3 IP-GDGT 80;4 IP-GDGT 80;5 P-GDGT 80;0 P-GDGT 80;2 P-GDGT 80;4 P-GDGT 80;5
92.3 3.94 3.0 2.25 0.3 0.17 0.2 0.07 0.1 0.10 0.5 0.37 0.2 0.13 2.2 1.16 0.1 0.06 0.1 0.05 0.2 0.05 0.7 0.19 0.1 0.04
Tetraether lipid
P-GDGT
a
The values represent average SD n = 3 of biological replicate. Three extraction replicates were made and two analytical replicates.
storage between extraction and analysis, which varied between the different samples, as well as potential inconsistencies of the analysis method. With all these factors involved, the question
the liposome formulation studies one predominant lipid species, the diether lipid with phosphorylated head group (92.3%) was identified (Table 1). One other phosphorylated diether lipid was
Table 2 Characterization of liposome formulations. Liposome formulation
Calcein concentration
Dia. (nm)a
EPC EPC:chol EPC:chol:TLE EPC:chol EPC:chol:TLE
5 mM 5 mM 5 mM 50 mM 50 mM
182.0 3.0 185.9 2.3 271.7 7.1 249.4 6.0 162.7 4.7
a
Values represent average SD n = 3 (three measurements of one typical liposome dispersion).
where the observed variability in lipid composition in this study comes from can't be finally answered. Compared to this, it seems as the extraction method only had minor influences on lipid composition. Based on these observations the DAC extraction method rather than the sonication method was chosen for further use because of its lower requirements in time and labor. Subsequently we scaled up the DAC method in order to extract 6 g of cell pellet per run and pooled the obtained lipid extracts for subsequent liposome preparation. In order to monitor the lipid composition, the pooled lipid extract was analyzed by shotgun lipidomics, with the results presented in Table 1. Within the pooled crude lipid extract used for
found, containing an inositol phosphate moiety (3.0%). The tetraether lipid species-fraction in the lipid extract amounted to 5% of the total lipid pool comprising 12 lipid species within four lipid classes. Like mentioned earlier, the lipid composition reported here differs quite substantially from our earlier results gained by small-scale extraction, where a higher amount of tetraether lipids was detected (Jensen et al., 2015a). The differences in the lipid profiles may be ascribed to the different lipid extraction methods, the cultivation conditions, inconsistencies in the analysis method or to the biological variability as described earlier. However, the high-resolution analysis of the crude lipid extract and the determination of the dominant lipid species will in the
Fig. 2. Comparison of loss of entrapped calcein from the three different liposome fomulations incubated with FaSSIF after 90 min at 37 C. Values represent average SD n = 3.
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Fig. 3. Release of entrapped calcein from the liposome formulation with ether lipids as a function of taurocholate concentration. Single values.
future help to verify how lipid composition and stability of liposomes are correlated. Obviously further investigations are needed to secure a lipid supply of more constant lipid composition. 3.2. Liposome characterization We compared three liposome formulations: pure egg PC, a mix of egg PC and cholesterol, and egg PC/cholesterol with the crude archaeal lipid extract. All three liposome formulations could be extruded without difficulties. The ether lipid liposomes showed a slightly larger diameter than the conventional liposomes (not containing ether lipids) (Table 2). However, for all three liposome formulations a reliable fractionation of free and entrapped marker could be observed. The fact that liposomes could be prepared from a mix of egg PC/cholesterol with 18% of crude lipid extract of S.
islandicus is in itself remarkable because earlier attempts had failed in this endeavor (Choquet et al., 1994). The only successful attempts to prepare mixed liposomes with lipids from the order Sulfolobales have used the purified polar lipid fraction E from S. acidocaldarius, which contains 3 tetraether compounds (Chang, 1994; Choquet et al., 1994) or a semi-synthetic tetraether lipid, GDNT with a modified head group (Parmentier et al., 2011a). 3.3. Loss of entrapped marker from liposomes upon exposure to bile salt Three selected liposome formulations, pure egg PC, egg PC with cholesterol and a formulation with ether lipids was initially incubated with FaSSiF medium (as described in Kloefer et al., 2010) in order to investigate if the liposomes formulation could survive
Fig. 4. Loss of entrapped calcein from the three liposome formulations after 90 min incubation at different taurocholate concentrations at 37 C. Entrapped calcein concentration 5 mM (A) or 50 mM (B). Values represent an average SD with n = 3, * = is a single experiment.
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intestinal conditions. Minor and comparable losses of calcein were observed for all three formulations (a loss of calcein from 6 to 12%) (Fig. 2). Thereby no significant differences were observed between the three formulations. All recovery values were in a range between 80% and 120%. The human intestinal fluid has a considerable variability in concentration of bile salts from 0.3 to 9.6 mM (for a recent review see Fuchs and Dressman, 2014). Having this range of bile salt concentrations in mind, we employed pure taurocholate up to more than double these concentrations in the stress tests. First the stability of the liposome formulation containing archaeal lipids was screened over a wide range of taurocholate concentrations. This pilot experiment showed that with increasing concentrations of taurocholate the loss of calcein from the liposomes increased from 6.1% (buffer as control) to 22.5% (4.4 mM), 92.9% (8.9 mM) to 98.9% (22 mM) (Fig. 3). Based on this result, the two other liposome formulations were also tested with concentrations of taurocholate up to 8.9 mM. In the control experiment with PBS there was no significant difference in the loss of calcein between the three formulations (Fig. 4A, p = 0.087). At taurocholate concentrations of 4.4 mM, the egg PC liposomes showed a considerable leakage of calcein, whereas from the two other liposome formulations almost no leakage occurred (Fig. 4A). At the highest taurocholate concentration tested (8.9 mM) a major loss of calcein was observed for all three liposome formulations. In the next step we raised the calcein load of the liposomes from 5 mM to 50 mM. With 50 mM calcein, the loss of calcein upon contact with taurocholate in general was higher than that from 5 mM calcein–liposomes, irrespective of liposome composition and taurocholate concentration (Fig. 4B). A reasonable explanation for this observation is a higher concentration gradient resulting in a higher diffusion potential. In addition we found that the osmolality for the two concentrations, 5 mM and 50 mM, was different (299.3 mOsm and 509.7 mOsm, respectively). Most likely a higher calcein load resulting in a higher osmolality inside the liposomes was triggering a more pronounced loss of encapsulated calcein. In conclusion, incorporation of crude archaeal lipid extract from S. islandicus in the liposome formulation had a stabilizing effect up to a taurocholate concentration of 4.4 mM and in FaSSIF medium as compared with liposomes made of egg PC. The stabilization effect was similar for the TLE liposomes and the egg PC:cholesterol liposomes. The crude lipid extract used for this experiment contained primarily diether lipids, suggesting that diether lipids from S. islandicus have a stabilizing effect. Previously it has been shown that archaeosomes made of 100% crude lipid extract from Thermoplasma acidophilum released in a comparable setting, i.e., upon 90 min incubation with 10 mM bile salt, almost all of the entrapped marker (Patel et al., 2000). In comparison, our mixed liposomes containing 18% crude lipid extract from S. islandicus showed similar stability against bile salts, however the exact lipid composition of the crude extract from T. acidophilum is unknown. Taken into account that it is very costly and labor-intensive to obtain and purify ether lipids, this knowledge is a step in the right direction in order to have an optimal and stable liposome formulation. However Fricker and colleagues have described that an egg PC-liposome formulation containing 9 mol% of a pure semisynthetic tetraether lipid (from S. acidocaldarius) had a stabilizing effect even at 10 mM taurocholate (>60% retained marker) at 90 min. This effect is superior to the stabilization we have observed, indicating that a single pure tetraether lipid exerts a stronger stabilizing effect to liposomes than the crude lipid extract investigated here. To employ a pure, semisynthetic tetraether lipid, however is costly and thus not suited for commercial production. In a previous study we have shown that the lipid composition of S. islandicus varies with culture conditions, especially a higher content of cyclopentane-moieties was observed with higher
culturing temperatures (Jensen et al., 2015b, in review). It could thus be investigated if crude lipid extracts obtained from S. islandicus grown under different culturing conditions would vary in their liposome stabilizing potential. It is also worth mentioning that we had observed higher percentages of tetraether lipids within crude lipid extracts from S. islandicus when we were extracting the cell pellet with a small-scale extraction method, which is suited for analytical purposes only. This result is more in line with the literature, where the main membrane lipids of S. islandicus are thought to be tetraether lipids (Jensen et al., 2015a). Set in perspective to the observation by Patel et al. (2000) that crude archaeal lipid extracts with predominantly tetraether lipids had a more pronounced stabilizing effect than those with predominantly diether lipids, there may be room for improvement with the crude lipid extract from S. islandicus. This gives rise to the hope that optimized culturing conditions as well as lipid extraction protocols can result in higher percentages of tetraether lipids at larger scale and thereby improve the stability of liposomes against bile salts. Finally, it should be mentioned, that there are several threats in the GI-tract, which the liposome formulation must resist. In this study we have focused on one threat only, the bile salts, since they have earlier been identified as the most important stressors for archaeosomes, where most of the entrapped marker was lost, in comparison to low pH and lipase activity (Patel et al., 2000). But since our ether liposome formulation only contains a smaller percentage of ether lipids it not known if the same will apply for this liposome formulation. Therefore it is relevant for future experiments to change the experimental settings by exposing the liposome formulation to more complex media containing both bile salts and lipases in order to simulate the intestinal conditions more appropriately. 4. Conclusion The current proof-of-concept study demonstrated that S. islandicus represents a potential source for biotechnological production of ether lipids. The total lipid extracts typically comprised both, diether- and tetraether-lipids in varying composition, as revealed by shotgun lipidomics. Egg PC-liposomes containing 18 mol% of the total lipid extract from S. islandicus were found to withstand in vitro exposure to bile salt (taurocholate) up to concentrations of 4 mM without substantial loss of the entrapped hydrophilic marker calcein. In contrast, conventional (egg PC) liposomes released almost their entire entrapped marker at this bile salts concentration, indicating that ether lipids from S. islandicus exert a liposome-stabilizing effect on liposomal drug carriers when it comes to bile salt-related stress. Future work will need to focus on increasing the tetraether lipid content in the crude lipid extract, studying if the recently described influence of culture conditions on ether lipid-composition and/or an optimized extraction process would yield even better membrane stabilizing effects. Finally, it remains to be investigated if these liposomestabilizing effects can be transferred into successful oral peptide delivery in vivo. Acknowledgements We thank Rachel Whitaker (University of Illinois) and Arnulf Kletzin (Technische Universität Darmstadt) for providing the Sulfolobus islandicus. We are grateful to Christer S. Ejsing, Renialdo Almeida, Albert Casanovas and Martin Hermansson for the scientific disscussion, data intepreation and help with mass spectrometry analysis. We thank Trine Johansson, Dina Skov and Tina Christiansen for technical assistance with cultivation and lipid extraction. We thank Askell Hinna for helpful discussions
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