Sandrine Cammas-Marion

New generation of liposomes called archaeosomes based on natural or synthetic archaeal lipids as innovative formulations for drug delivery.

Archaeosomes made from natural archaeal membrane lipids and/or synthetic lipid analogues have been extensively studied for potential applications in drug and vaccine delivery over the past decade only. Archaeal-type lipids consist of archaeol (diether) and/or caldarchaeol (tetraether) core structures wherein regularly branched and usually fully saturated phytanyl chains (20-40 carbons in lengths), are attached via ether bonds to the sn-2,3 carbons of the glycerol backbone. Archaeosomes constitute a novel generation of liposomes that exhibit high stabilities to low or high temperatures, acidic or alkaline pH, oxidative conditions, high pressure, action of phospholipases, bile salts and serum proteins. These properties associated with a good safety profile are beneficial for nanotechnological applications in drug and gene delivery. Additionally, archaeosome formulations could be used as efficient carriers of antigens and/or adjuvants promoting antigen-specific, humoral and cell-mediated immune responses, in addition to antigen-specific mucosal immune responses in the vaccinated hosts. The immune responses are well sustained over time, and are subject to strong memory responses. Nanodelivery-based vaccinations using archaeosomes could then represent a promising approach for treating and preventing infections, allergies, and neoplastic or cancer diseases. In this review, the few recent US, World and European patents developing archaeosomes for these biotechnological applications in Health are discussed.

Highly efficient gene transfer into hepatocyte-like HepaRG cells: new means for drug metabolism and toxicity studies

HepaRG progenitor cells are capable of differentiating into hepatocyte‐like cells that express a large set of liver‐specific functions. These cells, however, only express small amounts of an important cytochrome P450, the CYP2E1, which limits their use for toxicological studies of drugs metabolized by this pathway. Our aim was to establish an efficient transfection protocol to increase CYP2E1 expression in HepaRG cells. Transfection protocols of the green fluorescent protein (GFP) reporter gene were evaluated using electroporation and cationic lipids belonging to the lipophosphonates, lipophosphoramidates and lipids derived from glycine betaine. Following optimization of the charge ratios, plasmid DNA and formulations with neutral co‐lipids, the lipophosphoramidate compounds KLN47 and BSV10, allowed expression of the GFP in ∼50% of adherent progenitor HepaRG cells, while electroporation targeted GFP expression in ∼85% of both progenitor and differentiated cells in suspension. Transient enforced expression of active CYP2E1 was also achieved in progenitors and/or differentiated HepaRG cells using the electroporation and the lipophosphoramidate compound BSV10. Importantly, in electroporated cells, CYP2E1 expression level was correlated with a significant increase in CYP2E1‐specific enzymatic activity, which opens new perspectives for this CYP‐dependent drug metabolism and toxicity studies using HepaRG cells.

Folate‐Equipped Pegylated Archaeal Lipid Derivatives: Synthesis and Transfection Properties

We have previously shown that synthetic archaeal lipid analogues are useful vectors for drug/gene delivery. We report herein the synthesis and gene transfer properties of a series of novel di- and tetraether-type archaeal derivatives with a poly(ethylene glycol) (PEG) chain and further equipped with a folic acid (FA) group. The synthetic strategy and the purification by dialysis ensured complete removal of free FA. The lipids were mixed with a conventional glycine betaine-based cationic lipid and the resulting formulations were tested in transfection assays after complexation with plasmid DNA. All four novel co-lipids afforded efficient in vitro gene transfection. Moreover, the FA-equipped derivatives permitted ligand/receptor-based targeted transfection; their activity was inhibited when free FA was added to the transfection medium. These novel archaeal derivatives equipped with FA-PEG moieties may thus be of great interest for targeted in vivo transfection.

New macromolecular micelles based on degradable amphiphilic block copolymers of malic acid and malic acid ester

To study the behaviour of polymeric materials under in-vivo conditions, degradable macromolecular micelles based on amphiphilic block copolymers of poly(β-malic acid) as hydrophilic units and poly(β-malic acid alkyl esters) as hydrophobic blocks are studied. First three β-substituted β-lactones, benzyl malolactonate, butyl malolactonate, and butyl 3-methylmalolactonate were prepared, starting from aspartic acid. A prepolymer based on benzyl malate units was synthesized by anionic ring-opening polymerization of benzyl malolactonate. Then the carboxylic end groups of this prepolymer were used as initiator for the polymerization of the second lactone, e. g. butyl malolactonate or butyl 3-methylmalolactonate. The prepolymer and block copolymers have been characterized by 1H NMR and size exclusion chromatography (SEC). Degradable macromolecular micelles were prepared from the block copolymers by two different methods and characterized by dynamic light scattering and fluorescence measurements using pyrene as a fluorescence probe. It was shown that these amphiphilic degradable copolymers form stable micelles under physiological conditions (10–2M phosphate buffered solution, PBS, pH 7.4 with 0.15 M NaCl). Moreover, it was displayed that the characteristics of these macromolecular micelles, especially the critical micellar concentration (cmc), are depending on the chain length of both blocks and on the chemical structure of the hydrophobic block. A very important conclusion of this study is, that micelle formation is dependent on the pH of the medium. Therefore, besides the fact that such micelles are potentially degradable into non-toxic low molecular weight molecules, their properties and stability were proven to be pH-dependent. This property can lead development of an “intelligent” drug carrier able to release the entrapped biologically active molecule depending on the pH values.

Preparation and Characterization of Stealth Archaeosomes Based on a Synthetic PEGylated Archaeal Tetraether Lipid

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.

Synthesis and ITC characterization of novel nanoparticles constituted by poly(gamma-benzyl L-glutamate)-beta-cyclodextrin.

Imparting desired technological characteristics to polymeric nanoparticles requires the development of original polymers. In the present work, the synthesis and characterization of a novel PBLG‐derivative, the poly(γ‐benzyl L‐glutamate)‐β‐cyclodextrin (PBLG‐β‐CD‐50), have been carried out. Nanoparticles from either PBLG‐β‐CD‐50 polymer or from mixtures with PBLG have been prepared using a modified nanoprecipitation method. Spherically shaped nanoparticles with diameter in the range of 50–70 nm were obtained, as determined by dynamic laser light scattering and transmission electron microscopy. The presence of a surfactant in the suspension medium had almost no influence on these parameters and was not necessary to the shelf‐stability of the suspension. Further, isothermal titration microcalorimetry (ITC) experiments have been used to show unambiguously that about 20% of the cyclodextrins remain functional within the particles. Consequently, this system may be of interest when association of large amounts of hydrophobic drugs to nanoparticles is required. Copyright

Metal catalyzed ring-opening polymerization of benzyl malolactonate: a synthetic access to copolymers of β-benzyl malolactonate and trimethylene carbonate

The “immortal” coordination–insertion ring-opening polymerization of benzyl malolactonate (MLABe) initiated by the two-component catalyst system based on the zinc amide precursor, (BDI)Zn[N(SiMe3)2] (BDI = β-diiminate ligand), and benzyl alcohol (BnOH) acting as a co-initiator and a chain transfer agent proceeds in bulk at 40 °C. Functional telechelic poly(β-benzyl malolactonate)s, H-PMLABe-OBn, are thus obtained. Sequential copolymerization with trimethylene carbonate (TMC) affords block copolymers, PTMC-b-PMLABe, which are alternatively prepared from the chemical coupling of the PMLABe-COOH and PTMC-OH homopolymers. Simultaneous copolymerization of both the lactone and the carbonate monomers offers the PTMC-co-PMLABe random copolymers. The (co)polymers have been characterized by NMR, FT-IR, SEC and DSC analyses. These represent the first examples of β-benzyl malolactonate/carbonate copolymers. More importantly, these (co)polymers could be synthesized free of metallic residues thereby making them suitable as biomedical and pharmaceutical biomaterials.

PEGylated degradable composite nanoparticles based on mixtures of PEG-b-poly(γ-benzyl L-glutamate) and poly(γ-benzyl L-glutamate).

In the present work, the possibility to obtain PEGylated nanoparticles from two PBLG derivatives, PEG-b-poly(γ-benzyl L-glutamate), PBLG-PEG-60, and poly(γ-benzyl L-glutamate), PBLG-Bnz-50, by nanoprecipitation has been investigated. Particles were prepared not only from one polymer (PBLG-PEG-60 or PBLG-Bnz-50), but also from mixtures of two PBLG derivatives, PBLG-PEG-60 and PBLG-Bnz-50, in different ratios (90/10, 77/23, and 40/60 wt %). Because of the presence of PEG chains, hydrophilic particles were obtained, which was confirmed by ζ potential measurements (ζ from -13 mV and -21 mV) and by isothermal titration microcalorimetry (ITC). This last technique has shown no heat exchange when BSA was added to PEGylated nanoparticles. Further, complement activation has been evaluated by crossed immuno-electrophoresis demonstrating that the introduction of 77 wt % of PEGylated PBLG chains in the particles was enough to ensure a low complement activation activity. This effect was strongly correlated to the ζ potential of the particles, which decreased with an increase of PEG chains content. Interestingly, such properties are of interest for the preparation of degradable stealth nanocarriers. Moreover, it is suggested that the introduction of a reasonable amount (up to 20 wt %) of a second copolymer in the particle composition can be possible without modifying their stealth character. Moreover, the presence of this second copolymer would help to introduce a second functionality to the particles.

4-Alkyloxycarbonyl-2-oxetanones and 3-alkyl-4-alkyloxycarbonyl-2-oxetanones as versatile chiral precursors in the design of functionalized polyesters with a controlled architecture

The necessity for accessing a very large variety of high-molecular-weight racemic and optically active polyesters aimed at biomedical or chemical applications has led to the growth of a large series of racemic and optically active 4-alkyloxycarbonyl-2-oxetanones and 3-alkyl-4-alkyloxycarbonyl-2-oxetanones. Different synthesis routes have been established to prepare these monomers according to the bound ester group structure and the required enantiomeric or diastereomeric excess. The basic molecules were aspartic acid, malic acid or their 3-alkyl derivatives. The introduced ester group can be chiral, reactive, hydrophobic, hydrophilic or bioactive. The major interest in this enlarged monomer family lies in the possibility of have at ones disposal β-substituted β-lactones with very strict control of the stereogenic center configuration. This property is important with regard to the tailoring of well-defined polymeric materials. A second benefit lies in the tailor-making of complex architectures by the copolymerization of two or more β-substituted β-lactones. In al cases, the resulting materials have the composition of the monomer feed and a completely controlled configurational structure.

Folate PEGylated archaeal lipids: cell targeting and drug delivery.

Archaeosomes are of interest for potential biotechnological applications such as biomaterial delivery. Indeed, the archaeal lipid analogues improve the stability of delivery systems, including oral administration [1,2]. We developed new archaeal lipid analogues equipped with cell targeting ligands such as folic acid (FA) [3]. In vitro transfection efficiencies of formulations including these lipids were evaluated on several cell lines, and have shown good efficiencies at neutral charge ratios and a clear ligand mediated cell internalisation.