The present studies were focused on the formation and characterization of sterically stabilized archaeosomes made from a synthetic PEGylated archaeal lipid. In a first step, a synthetic archaeal tetraether bipolar lipid was functionalized with a poly(ethylene glycol), PEG, and (PEG45-Tetraether) with the aim of coating the archaeosome surface with a sterically stabilizing hydrophilic polymer. In a second step, Egg-PC/PEG45-Tetraether (90/10 wt%) archaeosomes were prepared, and their physicochemical characteristics were determined by dynamic light scattering (size, polydispersity), cryo-TEM (morphology), and by high-performance thin layer chromatography (lipid composition), in comparison with standard Egg-PC/PEG45-DSPE formulations. Further, a fluorescent dye, the carboxyfluorescein, was encapsulated into the prepared archaeosomes in order to evaluate the potential of such nanostructures as drug carriers. Release studies have shown that the stability of Egg-PC/PEG45-Tetraether-based archaeosomes is significantly higher at 37∘C than the one of Egg-PC/PEG45-DSPE-based liposomes, as evidenced by the slower release of the dye encapsulated into PEGylated archaeosomes. This enhanced stability could be related to the membrane spanning properties of the archaeal bipolar lipid as already described with natural or synthetic tetraether lipids.
In the drug-delivery field, several nanocarriers have been proposed to improve the therapeutic index of various biologically active molecules such as peptides. Indeed,
Within this context, archaeosomes, made with one or more of either the ether lipids found in Archaea bacteria or synthetic archaeal lipids, constitute a novel family of liposomes exhibiting higher stabilities in several conditions, such as high temperature, alkaline or acidic pH, presence of phospholipases, bile salts, and serum media [
Over the last decade, our research group has developed synthetic analogues of natural archaeal tetraether lipids and studied their uses in cationic archaeosome formulations as efficient gene delivery systems [
Egg-PC was purchased from Sigma. 1,2-distearoyl-
HPTLC plates (20*10 cm, silica gel 60, 0.2 mm layer thickness, Nano-Adamant UV254) were purchased from Macherey-Nagel. Before use, the HPTLC plates were prewashed with methanol, dried on a CAMAG TLC plate heater III at 120°C for 20 min, and kept in an aluminum foil in a desiccator at room temperature. All solvents were of HPTLC grade.
A mixture of tetraether diol
To a solution of alcohol
To a solution of carboxylic acid
Stock solutions of Egg-PC (1 mg/mL) and PEG45-DSPE (1 mg/mL) were prepared in CHCl3 : CH3OH (2 : 1, v/v), while stock solutions of PEG45-Tetraether (1 mg/mL) were prepared in CHCl3.
Liposomes and archaeosomes were obtained by the hydration method as already described elsewhere [
PEG45-Tetraether (90 : 10 wt%) based archaeosomes and Egg-PC/PEG45-DSPE (90 : 10 wt)
The size (average diameter obtained by the cumulant result method), polydispersity and zeta potential of the formulations were measured by dynamic light scattering using a Delsa Nano Beckman Coulter apparatus at 25°C. The samples were diluted 2 times with milliQ water.
The cryo-TEM analysis of PEGylated liposomes and PEGylated archaeosomes was realized by Dr. Olivier LAMBERT at the University of Bordeaux (Group “Chimie et Biologie des Membranes et Nano-objets”, UMR 5248 CNRS).
Each sample (5
The lipid compositions of formulations were determined after ultrafiltration. The samples were filtered through 10 000 NMWL pore filters (Micron YM-10, Millipore Corporation) by ultracentrifugation at 15 000 g for 1 hour at 15°C. The supernatants were recovered, lyophilized, dissolved in 1 mL of methanol, and analyzed by HPTLC using the automated HPTLC system from CAMAG (Muttenz, Switzerland). The samples, the appropriate lipid standard solutions and a blank solution composed by pure methanol were spotted on 20 × 10 cm HPTLC plates using the Automatic TLC Sampler 4 from CAMAG (Muttenz, Switzerland). Each lane was spotted 10 mm above the bottom edge of the plate and was 6 mm length with 17 mm spacing between lanes. The spotting volume was 10 Egg-PC PEG45-DSPE PEG45-Tetraether
Peak heights and peak areas were used for quantification. Calibration curves were calculated for each lipid or archaeal lipid, with a linear regression mode. In order to reduce experimental errors, individual calibration curves were obtained for every HPTLC plate. The amount of Egg-PC and PEG45-DSPE in liposomes, after ultrafiltration, and of Egg-PC and PEG45-Tetraether in archaeosomes, after ultrafiltration, were calculated from the calibration curves.
CF release profile from both PEGylated archaeosomes and PEGylated liposomes was measured by fluorescence using a Fluoromax-3 (Horiba) spectrofluorimeter with excitation and emission wavelengths of 490 and 515 nm, respectively. Release was studied at 4°C and 37°C. The fluorescence of both formulations was measured at T0, before (I0) and after (Imax) Triton-X-100 (2 v%) addition (total disruption of liposomial membranes) and at various times (It) until almost complete CF release at 4°C and at 37°C. Release of the incorporated dye was calculated using the following equation:
Archaeosomes made with one or more of the ether lipids found in Archaea represent an innovative family of liposomes that demonstrate higher stabilities to several conditions in comparison with conventional liposomes. The definition of archaeosomes also includes the use of synthetically derived lipids that have the unique structure characteristics of archaeobacterial ether lipids, that is, regularly branched phytanyl chains attached via ether bonds at
These atypical characteristics should be particularly useful for the preparation of highly stable archaeosomes. In particular, specific archaeal lipid membrane properties have to be considered in view to optimize the performance of archaeosomes: (1) the ether linkages are more stable than esters over a wide range of pH, and the branching methyl groups help both to reduce crystallization (membrane lipids in the liquid crystalline state at ambient temperature) and membrane permeability (steric hindrance of the methyl side groups); (2) the saturated alkyl chains would impart stability towards oxidative degradation; (3) the unusual stereochemistry of the glycerol backbone (opposite to mesophilic organisms) would ensure resistance to attack by phospholipases released by other organisms; (4) the bipolar lipids span the membranes and enhance their stability properties and (5) the addition of cyclic structures (in particular five-membered rings) in the transmembrane portion of the lipids appears to be a thermoadaptive response, resulting in enhanced membrane packing and reduced membrane fluidity.
Consequently, formulations including archaeal lipids demonstrate relatively higher stabilities to oxidative stress, high temperature, alkaline or acidic pH, action of phospholipases, bile salts, and serum media. Archaeosomes can be formed using standard procedures (hydrated film submitted to sonication, extrusion or detergent dialysis) at any temperature in the physiological range or lower, thus making it possible to encapsulate thermally labile compounds. Moreover, they can be prepared and stored in the presence of air/oxygen without any degradation. The
However, to study in depth archaeolipid structure-archaeosome property relationships with a view of optimizing the performance of these unusual liposomes as gene/drug nanocarriers, sufficient amounts of pure natural lipids are required. Well-defined lipids are difficult to isolate from natural extracts, and chemical synthesis appears, therefore, as an attractive means of producing model lipids that mimic the natural lipids. Within this context, our group focused on the synthesis and the evaluation of chemically pure archaeal diether and tetraether lipids that retain some of the essential structural features of archaeal membrane lipids. These studies clearly showed the interest in developing archaeosome technology from synthetic tetraether lipids possessing neutral, zwitterionic, or cationic polar heads groups for
In order to propose a stealth version of synthetic archaeosomes that could increase blood circulation longevity by reducing or preventing protein binding and/or by inhibiting cell binding/uptake, an additional archaeosome formulation based on a novel synthetic tetraether lipid was developed. These stealth archaeosomes could be suitable for the encapsulation and the
For that purpose, an archaeosome formulation composed by 90 wt% of a classical lipid, Egg-PC, and 10 wt% of a PEGylated tetraether archaeal lipid, PEG45-Tetraether (Figure
Structure of Egg-PC, PEG45-DSPE, and PEG45-Tetraether.
The novel PEGylated archaeal lipid (PEG45-Tetraether) was synthesized through the functionalization of the tetraether backbone at one terminal end. The synthesis of this unsymmetrical PEGylated lipid involved the monoprotection of the starting tetraether diol
Synthesis of PEG45-Tetraether lipid.
As described in the experimental part, formulations have been prepared using the classical lipid film hydration method followed by vesicle size reduction under sonication. The mean particle size and zeta potential of archaeosomes and liposomes were measured by dynamic light scattering. Particle mean diameters and polydispersity index are gathered in Table
Size (cumulant results), polydispersity (Ip), and zeta potential of prepared formulations. (ND = nondetermined).
Formulation | Size (nm), (Std Dev) | Ip | Zeta potential (mV) |
---|---|---|---|
Egg-PC/PEG45-DSPE | 70 (40) | 0.30 | −20.0 ± 9 |
Egg-PC/PEG45-Tetraether | 80 (30) | 0.26 | −13.0 ± 6 |
CF-encapsulated Egg-PC/PEG45-DSPE | 90 (37) | 0.21 | Nd |
CF-encapsulated Egg-PC/PEG45-Tetraether | 100 (45) | 0.26 | Nd |
Cryo-TEM was employed to investigate the morphology of the vesicles composed of PEGylated lipids. The images in Figure
Cryo-TEM photos of (a) Egg-PC/PEG45-Tetraether (90 : 10 wt%) archaeosomes and (b) Egg-PC/PEG45-DSPE (90 : 10 wt%) liposomes. Bar is 50 nm.
Besides these characteristics, it is of great interest to determine the lipid composition after formulation. For that purpose, we have used an innovative method based on quantitative thin layer chromatography, named high performance thin-layer chromatography (HPTLC). The HPTLC is a qualitative and quantitative analytical method allowing obtaining reproducible and reliable results [
Amounts of lipids contained in liposomes and archaeosomes calculated from HPTLC data. The given values are an average between peak height and peak area values. The values are reported to a volume of 1 mL.
Liposome formulations | Archaeosome formulations | ||
---|---|---|---|
Egg-PC | Initial amount ( | 0.900 (90 wt%) | 0.900 (90 wt%) |
PEG45-DSPE | Initial amount ( | 0.100 (10 wt%) | — |
— | |||
PEG45-Tetraether | Initial amount ( | — | 0.100 (10 wt%) |
— |
HPTLC measurements: (a) Scan of a plate at 366 nm (fluorescence mode); (b and c) standard curves, based on peak height, for each lipid composing the prepared liposomes and archaeosomes. (AU = arbitrary unit).
Scan of a plate at 366 nm (fluorescence mode)
EggPC/PEG45-DSPE (90 : 10 wt%) liposomes
EggPC/PEG45-Tetraether (90 : 10 wt%) archaeosomes
To assess vesicle stability, the kinetics of encapsulated CF release from PEG-bearing liposomes and archaeosomes was studied at 4°C (standard storage temperature of liposomal formulations) and 37°C (human physiological temperature). The percent release of CF was calculated from the formula described in the experimental part after evaluating the initial amount of encapsulated CF. Thus, a part of the sample containing the vesicle dispersion was treated with triton X-100 [
The release profile of CF from vesicles at 4°C (Figure
Release (%) of CF from Egg-PC/PEG45-Tetraether (90 : 10 wt%) archaeosomes and from Egg-PC/PEG45-DSPE (90 : 10 wt%) liposomes at (a) 4°C and (b) 37°C.
CF release at 4°C
CF release at 37°C
Despite their apparent identical characteristics in terms of morphology and surface potential, PEGylated liposomes and archaeosomes exhibited different vesicle stabilities. The presence of only 10 wt% of archaeal tetraether lipid in the liposomal formulations increased significantly the nano-object stability and allowed a slow release of the encapsulated dye at 37°C. This enhanced stability could result from the membrane spanning organization of the PEGylated tetraether lipids within the Egg-PC bilayer membrane, forming a monolayer as previously shown with synthetic cationic tetraethers [
In conclusion, we have demonstrated that small proportions of a novel synthetic PEGylated archaeolipid added to a liposomal formulation increase significantly the nanovector stability and slow down the constant dye release at 37°C. This result is quite promising in so far as a similar behavior could be expected for
The authors would like to thank the partners of the project Sealacian for valuable discussion. They also would like to thank the CNRS, the Direction Générale des Entreprises (DGE), the Région Bretagne, and the Ministère de l’Enseignement Supérieur et de la Recherche for financial support. Finally, the authors thank Dr. Olivier Lambert for cryo-TEM analysis. J. Barbeau would like to thank the DGE for the financial support, which enabled her to achieve this study.