Philippe Pierrat

Efficient in vitro and in vivo pulmonary delivery of nucleic acid by carbon dot-based nanocarriers

Cationic carbon dots were fabricated by pyrolysis of citric acid and bPEI25k under microwave radiation. Various nanoparticles were produced in a 20-30% yield through straightforward modifications of the reaction parameters (stoichiometry of the reactants and energy supply regime). Particular attention was paid to the purification of the reaction products to ensure satisfactory elimination of the residual starting polyamine. Intrinsic properties of the particles (size, surface charge, photoluminescence and quantum yield) were measured and their ability to form stable complexes with nucleic acid was determined. Their potential to deliver plasmid DNA or small interfering RNA to various cell lines was investigated and compared to that of bPEI25k. The pDNA in vitro transfection efficiency of these carbon dots was similar to that of the parent PEI, as was their cytotoxicity. The higher cytotoxicity of bPEI25k/siRNA complexes when compared to that of the CD/siRNA complexes however had marked consequences on the gene silencing efficiency of the two carriers. These results are not fully consistent with those in some earlier reports on similar nanoparticles, revealing that toxicity of the carbon dots strongly depends on their protocol of fabrication. Finally, these carriers were evaluated for in vivo gene delivery through the non-invasive pulmonary route in mice. High transgene expression was obtained in the lung that was similar to that obtained with the golden standard formulation GL67A, but was associated with significantly lower toxicity. Post-functionalization of these carbon dots with PEG or targeting moieties should significantly broaden their scope and practical implications in improving their in vivo transfection efficiency and biocompatibility.

Well-defined star shaped polymer-fullerene hybrids via click chemistry†

The use of a hexakisazido macrocyclic methanofullerene proves to be highly efficient in the preparation of 6-arm star polymers via copper(I) catalyzed azide-alkyne cycloaddition.

Di‐ and Dodeca‐Mitsunobu Reactions on C60 Derivatives: Post‐Functionalization of Fullerene Mono‐ and Hexakis‐Adducts

The diverse addition patterns of fullerene adducts make them attractive molecules, generating widespread application. Firstly, in terms of the latter, mono-adducts are extensively used in opto-electronic devices, owing to the fullerene core s capacity to accept up to six electrons. A second important application involves the utilization of fullerene adducts as structural components in material sciences. With their octahedral topology, Th-symmetric hexakis-adducts, are particularly intriguing. Amongst others, the latter have been used in C60-based star polymers. [5] In the aforementioned contexts, the relatively stable methanofullerene adducts obtained through Bingel cyclopropanation reactions are of particular interest. However, this functionalization approach—primarily developed by A. Hirsch and later optimized by Y.-P. Sun—suffers from two critical weaknesses. One is its effectively exclusive limitation to malonates; only a few exceptions are known. The other is a structural concern. In fact, quite often only the use of fairly simple malonates leads to the desired fullerene adducts in reasonable yields without tedious purifications. In order to overcome these problems, several groups have developed post-functionalizations of methanofullerene adducts. For instance, J.F. Nierengarten adapted the copper-mediated Huisgen 1,3dipolar cycloaddition reaction and applied it to the preparation of complex hexasubstituted fullerenes. A. Hirsch developed a selective deprotection–functionalization sequence of fullerene e,e,e-trisadducts. Our own group derivatized hexakis methanofullerenes by using several organometallic cross-coupling reactions. This article outlines the use of the Mitsunobu reaction as an efficient tool for the post-functionalization of fullerene monoand hexakis-adducts. The mild, virtually neutral conditions under which this dehydrative coupling of an alcohol with a pronucleophile (generally an acid) proceeds, prompted our group to use this reaction for the derivatization of fullerene adducts. To this end, we have prepared a C60 mono-adduct bearing 2 hydroxyl groups and a hexakisadduct with 12 hydroxyl functions. The preparation of hexakis compound 4 is depicted in Scheme 1. Ethylene glycol was mono-protected as tert-butyldimethylsilyl ether according to a previously reported procedure. Subsequent diesterification of malonic acid was performed in the presence of DCC in 97% yield. Hexakis-adduct 3 was then readily synthesized by using the optimized conditions

A Cationic Phospholipid–Detergent Conjugate as a New Efficient Carrier for siRNA Delivery

Nucleic acids have a huge intrinsic therapeutic potential that, however, relies heavily on the development of methods for their delivery to their intracellular target site. Cationic lipids and polymers were introduced more than twenty years ago for the delivery of DNA molecules to cells. Since then, they have become useful tools for biomedical research and have also been adopted for siRNA delivery. Their mode of action has been roughly determined. It is based on the property of adherent cell lines to easily internalize large quantities of cationic complexes in endocytic compartments, and on the incorporation of membrane-perturbing elements within internalized complexes to trigger subsequent release of the nucleic acid payload into the cytosol. Cationic lipids forming hexagonal phase or formulations of cationic lipids with the fusogenic lipid 1,2-dioleoyl-snglycero-3-phosphoethanolamine (DOPE) enable both the formation of cationic nucleic acid complexes (lipoplexes) and rupture of the endosome membrane. Yet, the endosomolytic activity of the current effective synthetic nucleic acid delivery systems remains low and effective delivery is only achieved by using cell entry pathways with a high flow rate. The membrane-disruptive properties of detergents have been considered for improving nucleic acid delivery. However, the use of detergents remains delicate because direct plasma membrane permeation might have an irreversible impact on cell viability. Cationic detergents have been shown to provoke DNA condensation but the resulting lipoplexes have turned out to be inactive or only poorly active, in vitro, when particles are prepared from cationic detergent mixed with DOPE. This is explained by the rapid exchange of the detergent molecules between the complex and the aqueous environment, which results in an irreversible decondensation of the nucleic acid even before entry into the cells. On the other hand, preparation of DNA lipoplexes from cationic lipids in the presence of Tween-80, a non-ionic detergent, has led to systems with enhanced gene transfection properties both in vitro and in vivo. These results led us to explore the consequences on nucleic acid delivery of the conjugation of a detergent molecule to a cationic lipid so that the detergent cannot be depleted from the transfection particle. We selected Triton X-100 (TX100) for its well-known detergent properties and for synthetic convenience. Furthermore, TX100 does not solubilize cholesterol-enriched raft domains, which might be involved in cationic lipoplexes internalization. TX100 was covalently linked to the phosphate group of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a major component of biological membranes (Figure 1). Diacylglycerophosphocholines (PCs) are normal cellular metabolites and offer the opportunity to easily generate cationic lipids by O-alkylation of the phosphate group. Gorman et al. first demonstrated that a PC-derived phosphotriester (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, EDMPC) can mediate efficient gene transfer. Later on, derivatives of DOPC (i.e., ethyl-DOPC, EDOPC, Figure 1) and other PCs were developed by MacDonald et al. , the physical and transfection properties of which were extensively investigated. We hypothesized that the covalent attachment of a detergent molecule and a natural phospholipid to form a cationic phospholipid–detergent conjugate might improve cell uptake and transfection efficiency of lipoplexes prepared thereof. TX100 conjugation to DOPC was realized according to Scheme 1. Detergent activation with trifluoromethanesulfonyl anhydride provided the corresponding sulfonyl ester. This compound is unstable and decomposes in a few hours on standing at room temperature in chloroform. Analysis of degradation products is consistent with a deoligomerization process leading to the formation of dioxane as reported previously with other PEG derivatives. Consequently, the sulfonyl ester (1 equiv) was directly submitted to nucleophilic displacement by the anionic phosphate in DOPC (3 equiv). Conjugate 1 was obtained in 32% yield and unreacted DOPC (61%) was recovered after purification. The use of a larger excess of activated TX100 (7 equiv) improved the DOPC conversion but finally proved detrimental as the purification of the product became difficult (1: 22%; recovered DOPC: 24%). Control compound 2 was prepared similarly, by replacing Triton X-100 by polyethylene glycol mono[a] Dr. P. Pierrat, Dr. G. Creusat, Dr. G. Laverny, Prof. F. Pons, Dr. G. Zuber, Dr. L. Lebeau Laboratoire de Conception et Application de Mol!cules Bioactives CNRS, Universit! de Strasbourg, Facult! de Pharmacie 74 Route du Rhin, BP 60024, Illkirch (France) Fax: (+33)333-6885-4306 E-mail : llebeau@unistra.fr Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201103645.

Phospholipid–Detergent Conjugates as Novel Tools for siRNA Delivery

One of the potential benefits of drug delivery systems in medicine is the creation of nanoparticle-based vectors that deliver a therapeutic cargo in sufficient quantity to a target site to enable a selective effect, width of the therapeutic window depending on the toxicity of the vector and the cargo. In this work, we intended to improve the siRNA delivery efficiency of a new kind of nucleic acid carrier, which is the result of the conjugation of the membrane phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) to the membrane-active species Triton X-100 (TX100). We hypothesized that by improving the biodegradability the cytotoxicity of the conjugate might by reduced, whereas its original transfection potential would be tentatively preserved. DOPC was conjugated to Triton X-100 through spacers displaying various resistance to chemical hydrolysis and enzyme degradation. The results obtained through in vitro siRNA delivery experiments showed that the initial phosphoester bond can be replaced with a phospho(alkyl)enecarbonate group with no loss in the transfection activity, whereas the associated cytotoxicity was significantly decreased, as assessed by metabolic activity and membrane integrity measurements. The toxicity of the conjugates incorporating a phospho(alkyl)enesuccinnate moiety proved even lower but was clearly balanced with a reduction of the siRNA delivery efficiency. Hydrolytic stability and intracellular degradation of the conjugates were investigated by NMR spectroscopy and mass spectrometry. A general trend was that the more readily degraded conjugates were those with the lower toxicity. Otherwise, the phospho(alkyl)enecarbonate conjugates revealed some hemolytic activity, whereas the parent phosphoester did not. The reason why these conjugates behave differently with respect to hemolysis might be a consequence of unusual fusogenic properties and probably reflects the difference in the stability of the conjugates in the intracellular environment.

A study of insoluble monolayers by deposition at a bubble interface

We propose an experimental approach, alternative to Langmuir trough, pendant or sessile drop experiments, to study insoluble monolayers at the air–water interface. The method is based on the direct deposition of an insoluble surfactant at the interface of an air bubble, measuring the surface tension according to the classical axisymmetric drop shape analysis (ADSA) technique. This bubble configuration, in contrast to the classical ones for studying Langmuir monolayers have several remarkable advantages like the easy control of the surrounding bulk composition (opening new potential research applications), the fast experimental time for a monolayer to be ready (<20 min), the small bulk volume (10 mL), and mostly the simple way to carry out dilatational rheology. The experiment consists of performing compression of an insoluble monolayer recording the Π–AB curve (Π is the interfacial pressure and AB the bubble area) and obtaining dilatational rheology over the compression range establishing the E–Π curve (with E is the elastic modulus). We showed that the experimental results can be satisfactorily fitted using the Volmers equation of state including the two-dimensional compressibility factor e, offering access to the excluded area per molecule ω0 and to the number N of molecules at the interface, without initially knowing the amount of deposited material. This proof of concept study was carried out on dioleoyl-sn-glycerophosphatidylcholine (DOPC), dipalmitoyl-sn-glycerophosphatidylcholine (DPPC), and cholesterol at 20 °C, systems chosen to show qualitative differences in their thermodynamic behavior upon monolayer compression. Likewise, dilatational rheology of these insoluble monolayers allows evidencing the compressibility of the DOPC monolayer in contrast to the DPPC monolayer, and finally, the compression domains where the interface loses the surfactant through a comparison of the dilatational elasticity with the Gibbs elasticity calculated from the compression curves. Finally, we propose an example of the new application offered by the possibility to exchange the fluid phase surrounding the bubble, herein to study mixed monolayers made with soluble/insoluble surfactants (Tween 80/Span 65).

Bioresponsive Deciduous-Charge Amphiphiles for Liposomal Delivery of DNA and siRNA

ABSTRACTPurposeBiolabile cationic lipids were developed for efficient intracellular delivery of DNA and siRNA.MethodsThe compounds have been designed starting from the membrane lipid DOPC in a way they may loose their cationic charge when exposed to an acidic and/or enzymatic stimulus, such as those met during the journey of a lipoplex in biological media.ResultsThey demonstrated remarkable efficiency to deliver DNA in various cell lines (BHK-21, Calu-3, NCI-H292, and A549), with no significant cytotoxicity. Furthermore, two of the compounds (carbonate-based DOPC derivatives) revealed able to deliver small interfering RNA in U87Luc and A549Luc cancer cells and to mediate a selective 70–80% knockdown of the stably transfected luciferase gene.ConclusionsThe results show that the described bioresponsive cationic lipids have high DNA and siARN delivery activity which is encouraging in view of delivering a therapeutic nucleic acid to pulmonary tissues in vivo.

A Hexakis Terpyridine-Fullerene Ligand in Six-Fold Ruthenium, Iridium, and Iron Complexes: Synthesis and Electrochemical Properties.

An unprecedented straightforward route to six-fold terpyridine ligands around C60 , the latter being regioselectively functionalized in pseudo-octahedral positions using a six-fold Bingel reaction, is reported. Ruthenium, iridium, and iron complexes have been synthesized, and unambiguously characterized by NMR, MS, and cyclic voltammetry.

DOPC-Detergent Conjugates: Fusogenic Carriers for Improved In Vitro and In Vivo Gene Delivery

Phospholipid-detergent conjugates are proposed as fusogenic carriers for gene delivery. Eleven compounds are prepared and their properties are investigated. The ability of the conjugates to promote fusion with a negatively charged model membrane is determined. Their DNA delivery efficiency and cytotoxicity are assessed in vitro. Lipoplexes are administered in the mouse lung, and transgene expression Indeterminate inflammatory activity are measured. The results show that conjugation of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with C12 E4 produces a carrier that can efficiently deliver DNA to cells, with negligible -associated toxicity. Fusogenicity of the conjugates shows good correlation with in vitro transfection efficiency and crucially depends on the length of the polyether moiety of the detergent. Finally, DOPC-C12 E4 reveals highly potent for in vivo DNA delivery and favorably compares to GL67A, the current golden standard for gene delivery to the airway, opening the way for further promising developments.

Cationic DOPC-Detergent Conjugates for Safe and Efficient in Vitro and in Vivo Nucleic Acid Delivery.

The ability of a nonviral nucleic acid carrier to deliver its cargo to cells with low associated toxicity is a critical issue for clinical applications of gene therapy. We describe biodegradable cationic DOPC–C12E4 conjugates in which transfection efficiency is based on a Trojan horse strategy. In situ production of the detergent compound C12E4 through conjugate hydrolysis within the acidic endosome compartment was expected to promote endosome membrane destabilization and subsequent release of the lipoplexes into cytosol. The transfection efficiency of the conjugates has been assessed in vitro, and associated cytotoxicity was determined. Cellular uptake and intracellular distribution of the lipoplexes have been investigated. The results show that direct conjugation of DOPC with C12E4 produces a versatile carrier that can deliver both DNA and siRNA to cells in vitro with high efficiency and low cytotoxicity. SAR studies suggest that this compound might represent a reasonable compromise between the membrane activity of the released detergent and susceptibility of the conjugate to degradation enzymes in vitro. Although biodegradability of the conjugates had low impact on carrier efficiency in vitro, it proved critical in vivo. Significant improvement of transgene expression was obtained in the mouse lung tuning biodegradability of the carrier. Importantly, this also allowed reduction of the inflammatory response that invariably characterizes cationic‐lipid‐mediated gene transfer in animals.