Guy Zuber

Proton Sponge Trick for pH-Sensitive Disassembly of Polyethylenimine-Based siRNA Delivery Systems

Small interfering RNAs offer novel opportunities to inhibit gene expression in a highly selective and efficient manner but depend on cytosolic translocation with synthetic delivery systems. The polyethylenimine (PEI) is widely used for plasmid DNA transfection. However, the water-soluble PEI does not form siRNA polyplexes stable enough in extracellular media for effective delivery. We previously showed that rendering PEI insoluble in physiological media, without modifying drastically its overall cationic charge density, by simple conjugation with natural hydrophobic alpha-amino acids, can lead to effective siRNA delivery in mammalian cells. In here, we comprehensively investigated the mechanism behind the excellent efficacy of the leading PEIY vector. Our data revealed that the underlining proton sponge property is key to the effectiveness of the tyrosine-polyethylenimine conjugate as it may allow both endosomal rupture and siRNA liberation via an optimal pH-sensitive dissolution of the PEIY self-aggregates. Altogether, these results should facilitate the development of novel and more sophisticated siRNA delivery systems.

MiR-20a regulates ASK1 expression and TLR4-dependent cytokine release in rheumatoid fibroblast-like synoviocytes

Objective To evaluate whether miR-20a belonging to the cluster miR-17–92 is a negative regulator of inflammation in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) by modulating expression of apoptosis signal-regulating kinase (ASK) 1, a key component of the toll-like receptors 4 pathway, upstream of p38 mitogen-activated protein kinase. Methods Evaluation of miR-20a and ASK1 mRNA was performed by RT-qPCR. ASK1 protein expression was assessed by western blotting. Overexpression of miR-20a was performed by transfection of RA FLS and THP-1 cells with miR-20a mimics. Interleukin (IL)-6, CXCL-10, IL-1β and TNF-α release were measured by ELISA. The role of miR-20a in vivo was assessed by IL-6 release from macrophages obtained from mice injected intraperitoneally with vectorised miR-20a mimics. Results We showed that stimulation of RA FLS with lipopolysacharide (LPS) and bacterial lipoproteins (BLP) induces a drop in expression of miR-20a and that this decrease is associated with an upregulation of ASK1 expression. Using transfection of Ask1 3′UTR reporters, we demonstrate that Ask1 is a direct target of miR-20a. Overexpression of miR-20a led to a global decrease in ASK1 protein in BLP- and LPS-activated cells indicating that miR-20a regulates the expression of ASK1 at the translational level. Transfection of miR-20a mimics decreases IL-6 and CXCL10 release by RA FLS and IL-1β and TNF-α by activated THP-1 cells but only in response to LPS. Last, injection of vectorised miR-20a mimics to mice led to a global decrease in ASK1 protein expression and IL-6 secretion in LPS-activated macrophages. Conclusions Our data point toward an important role for miR-20a in the regulation of pro-inflammatory cytokines release, by controlling ASK1 expression in RA FLS.

Iodinated α-tocopherol nano-emulsions as non-toxic contrast agents for preclinical X-ray imaging

Micro-computed tomography (micro-CT) is an emerging imaging modality, due to the low cost of the imagers as well as their efficiency in establishing high-resolution (1-100 μm) three-dimensional images of small laboratory animals and facilitating rapid, structural and functional in vivo visualization. However use of a contrast agent is absolutely necessary when imaging soft tissues. The main limitation of micro-CT is the low efficiency and toxicity of the commercially available blood pool contrast agents. This study proposes new, efficient and non-toxic contrast agents for micro-CT imaging. This formulation consists of iodinated vitamin E (α-tocopheryl 2,3,5-triiodobenzoate) as an oily phase, formulated as liquid nano-emulsion droplets (by low-energy nano-emulsification), surrounded by a hairy PEG layer to confer stealth properties. The originality and strength of these new contrast agents lie not only in their outstanding contrasting properties, biocompatibility and low toxicity, but also in the simplicity of their fabrication: one-step synthesis of highly iodinated oil (iodine constitutes 41.7% of the oil molecule weight) and its spontaneous emulsification. After i.v. administration in mice (8.5% of blood volume), the product shows stealth properties towards the immune system and thus acts as an efficient blood pool contrast agent (t(1/2) = 9.0 h), exhibiting blood clearance following mono-exponential decay. A gradual accumulation predominantly due to hepatocyte uptake is observed and measured in the liver, establishing a strong hepatic contrast, persistent for more than four months. To summarize, in the current range of available or developed contrast agents for preclinical X-ray imaging, this agent appears to be one of the most efficient.

Self-Assembling Polyethylenimine Derivatives Mediate Efficient siRNA Delivery in Mammalian Cells

Synthetic 21–22-nt-long RNA duplexes (siRNAs) can trigger specific mRNA degradation and selectively control gene expression, and because of this they have potential therapeutic applications. Unfortunately, the intracellular localization of siRNA targets and the inability of oligonucleotides to diffuse across cellular membranes requires siRNA conjugation or formulation with synthetic delivery vectors. Interestingly, polyACHTUNGTRENNUNGethylenimine (PEI), a popular gene transfection agent that showed promise for cancer gene therapy, appears inefficient for siRNA delivery in vitro. The poor performance of PEI could be linked with the length of the siRNA anionic segment, which is too short to maintain electrostatic cohesion with the soluble cationic polymer. Consequently, siRNA polyplexes break apart too readily upon contact with polyanions present at cell surfaces. We reasoned that the transformation of water-soluble PEI into a molecule with self-assembly properties should stabilize siRNA polyplexes and hence favor overall siRNA delivery. A water-soluble polymer could acquire new aggregating properties through the addition of hydrophobic domains. We avoided using alkyl chains, which, by mixing with the cell lipid bilayer, might act as a detergent and cause cell death. Our preference was instead for natural a-amino acids with decreasing hydropathy indices, such as leucine (L), phenylalanine (F), tryptophan (W), and tyrosine (Y). Indeed, these amino acids constitute the cores of globular proteins and help maintain protein structures. They also can be coupled to PEI amines without destroying nucleic acid binding properties, because over the course of the reaction a cationic amine replaces the reacted one. Commercially available 25 kDa branched PEI contains primary, secondary, and tertiary amines in a ratio of 1:1:1. The primary amines were allowed to react fully with the succinimidyl esters of butyloxycarbonyl-protected amino-acids (Bocaa-OSu) (Scheme 1). Removal of the Boc groups with trifluoroacetic acid (TFA) and subsequent dialysis in aqueous HCl gave the desired products, with a-amino acid contents of 30 % per ethylenimine, in 30–60 % overall yields. The aggregating properties of the polymers were evaluated by measurement of dynamic light scattering (Table 1). All polymers, as hydrochloride salts, were soluble in water, even at a concentration of 0.5 m. Upon dilution in RPMI cell culture medium, no light scattering signals were detected in the cases of PEI and PEIL. On the other hand, PEIF, PEIW, and PEIY self-assembled into particles with apparent diameters between 0.3– 0.7 mm. Addition of siRNA permitted PEI and PEIL to form complexes and led to slight increases in the sizes of the self-assembling polymers in a range that had previously been observed to be effective for PEI-mediated gene delivery. Transmission electron micrographs showed that PEIY (Figure 1 A) forms fiber-like structures of fused irregular spheroids roughly 20 nm in diameter. Addition of siRNA (Figure 1 B) led to a twofold ACHTUNGTRENNUNGdiminution in the fiber diameters and to further clustering. Nanoparticles, including viruses, are too large to diffuse freely across membranes and thus need to divert the cell machinery for translocation. They first anchor to receptors on the cellular surface and are then actively dragged into cells within membrane-coated vesicles (often endosomes). Evidence suggests that the high buffer capacity of PEI enables rupture of the endosome membranes and hence effective nucleic acid translocation. We therefore examined whether amino acid modification impacted this important function by acid–base ACHTUNGTRENNUNGtitration (Figure 1). Profiles of modified PEIs superposed almost perfectly in the pH 6.0–8.0 range and showed that the polymers have higher buffering capacities than PEI, probably due to the electron-withdrawing effect of carboxamide on the aterminal amines. This greater “proton sponge” ability should Scheme 1. Synthesis. a) Boc-aa-OSu. b) TFA.

Polyethylenimine-carbon nanotube nanohybrids for siRNA-mediated gene silencing at cellular level

Carbon nanotubes (CNTs) covalently modified with low molecular weight polyethylenimine (PEI) are able to bind and deliver siRNA to cells with higher efficacy than a reference lipidic carrier. The performances of the nanohybrid are rationalized by the combination of the cell penetration and endosomal escape properties of CNTs and PEI, respectively.

Suppression of cervical carcinoma cell growth by intracytoplasmic codelivery of anti-oncoprotein E6 antibody and small interfering RNA.

Cervical cancer is caused by high-risk types of human papillomaviruses (HPV) that encode the E6 and E7 oncogenes. Silencing of E6 gene expression in HPV-positive cell lines by transfection of small interfering RNA (siRNA) with cationic lipids restores the dormant p53 tumor suppressor pathway. Because cationic lipids can also be used for intracytoplasmic delivery of proteins, we tested whether the delivery of monoclonal antibodies that bind to HPV16 E6 and neutralize its biological activity in vitro could restore p53 function in tumor cells. Here, we show that the 4C6 antibody is efficiently delivered into the cell cytoplasm using a lipidic reagent used for siRNA transfection. The delivery of 4C6 resulted in the nuclear accumulation of p53 protein in CaSki and SiHa cells but not in HeLa cells. Furthermore, the antibody-mediated p53 response was dramatically increased when a peptide corresponding to the 4C6 epitope and bearing a COOH-terminal cysteine residue was added to the transduction mixture. We found that a fraction of the added peptides were dimers that allowed the formation of antibody polymers adsorbed onto the lipidic matrix. With this system, the proliferation of CaSki and SiHa cells was strongly diminished, but no apoptosis was detectable. Remarkably, cell growth was almost totally suppressed by the addition of E6-specific siRNA to the transduction complex. The results indicate that the activity of E6 oncoprotein can be down-regulated in vivo by lipid-mediated antibody delivery and that antibodies and siRNA act synergistically when codelivered. This novel targeting strategy is simple to implement and may find therapeutic applications. [Mol Cancer Ther 2007;16(5):1728–35]

Contrast agents for preclinical targeted X-ray imaging

Micro-computed tomography (micro-CT) is an X-ray based instrument that it is specifically designed for biomedical research at a preclinical stage for live imaging of small animals. This imaging modality is cost-effective, fast, and produces remarkable high-resolution images of X-ray opaque skeleton. Administration of biocompatible X-ray opaque contrast agent allows delineation of the blood vessels, and internal organs and even detection of tumor metastases as small as 300 μm. However, the main limitation of micro-CT lies in the poor efficacy or toxicity of the contrast agents. Moreover, contrast agents for micro-CT have to be stealth nanoparticulate systems, i.e. preventing their rapid renal clearance. The chemical composition and physicochemical properties will condition their uptake and elimination pathways, and therefore all the biological fluids, organs, and tissues trough this elimination route of the nanoparticles will be contrasted. Furthermore, several technologies playing on the nanoparticle properties, aim to influence these biological pathways in order to induce their accumulation onto given targeted sites, organs of tumors. In function of the methodologies carried out, taking benefit or not of the action of immune system, of the natural response of the organism like hepatocyte uptake or enhanced permeation and retention effect, or even accumulation due to ligand/receptor interactions, the technologies are called passive or active targeted imaging. The present review presents the most recent advances in the development of specific contrast agents for targeted X-ray imaging micro-CT, discussing the recent advance of in vivo targeting of nanoparticulate contrast agents, and the influence of the formulations, nature of the nanocarrier, nature and concentration of the X-ray contrasting materials, effect of the surface properties, functionalization and bioconjugation. The pharmacokinetic and versatility of nanometric systems appear particularly advantageous for addressing the versatile biomedical research needs. State of the art investigations are on going to propose contrast agents with tumor accumulating properties and will contribute for development of safer cancer medicine having detection and therapeutic modalities.

Dependence of the cellular internalization and transfection efficiency on the structure and physicochemical properties of cationic detergent/DNA/liposomes.

Control of the structure and physicochemical properties of DNA complexed with nonviral vectors is essential for efficient biodistribution and gene delivery to cells. Cationic liposomes interact with DNA giving transfection competent but large and heterogeneous aggregates. On the other hand, cationic detergents condense DNA into small homogeneous but reversible complexes inefficient for transfection.

Pyridylthiourea-grafted polyethylenimine offers an effective assistance to siRNA-mediated gene silencing in vitro and in vivo

Success of synthetic interfering nucleic acids (siRNAs)-based therapy relies almost exclusively on effective, safe and preferably nanometric delivery systems which can be easily prepared, even at high concentrations. We prepared by chemical synthesis various self-assembling polymers to entrap siRNAs into stable polyplexes outside cells but with a disassembly potential upon sensing endosomal acidity. Our results revealed that pyridylthiourea-grafted polyethylenimine (πPΕΙ) followed the above-mentioned principles. It led to above 90% siRNA-mediated gene silencing in vitro on U87 cells at 10 nM siRNA concentration and did not have a hemolytic activity. Assembly of siRNA/πPΕΙ at high concentration was then studied and 4.5% glucose solution, pH 6.0, yielded stable colloidal solutions with sizes slightly below 100 nm for several hours. A single injection of these concentrated siRNA polyplexes into luciferase-expressing human glioblastoma tumors, which were subcutaneously xenografted into nude mice, led to a significant 30% siRNA-mediated luciferase gene silencing 4 days post-injection. Our results altogether substantiate the potential of self-assembling cationic polymers with a pH-sensitive disassembly switch for siRNA delivery in vitro and also in vivo experiments.

Online Synthesis of Diblock Cationic Oligonucleotides for Enhanced Hybridization to their Complementary Sequence

Oligonucleotides are finding an extremely large number of applications in molecular biology and diagnostics, and might become a very selective class of drugs for the treatment of a vast palette of diseases. Oligonucleotides are polyanions that exert their specific activity following hybridization to a complementary sequence borne by another polyanionic nucleic acid. As drug candidates, they must also be capable of crossing the anionic cell membrane. Simple electrostatic considerations imply that hybridization energy and cell binding could benefit from the addition of cationic groups to the oligonucleotide structure. To reach this goal, many synthetic approaches for introducing ammonium or guanidinium residues into oligonucleotides have been explored: phosphate backbone replacement, ribose or nucleic base modification and end conjugation of a polycation. However, hybridization specificity, nucleic acid-processing enzyme activity and metabolite toxicity concerns all point to the diblock approach, in which the polycation is appended to an otherwise natural oligonucleotide, as the best solution. Unfortunately, stepwise automated synthesis of oligonucleotide–cationic peptide conjugates is not yet routine. Unfortunately, conjugation chemistry between preformed large blocks is not straightforward, especially in water, where “super” zwitterions raise intractable solubility, purification and characterization problems. Moreover, molecular biology and diagnostics applications require fast and straightforward synthesis of any given base sequence linked to any ACHTUNGTRENNUNGorganic cation length. With these considerations as guidelines, we have focused our efforts towards developing a high-yield automated synthesis of oligonucleotide–oligocation conjugates that makes exclusively use of classical phosphoramidite chemistry. In addition to the four A-, T-, Gand C-protected trityl phosphoramidite vials, a linear polyamine N-protected trityl phosphoramidite vial was plugged into the oligonucleotide synthetizer machine to allow online, computer-driven, synthesis (Scheme 1). Decamer oligonucleotides bearing a global charge of up to +9 were synthesized in good yield and characterized by ESMS and PAGE. Duplex melting temperature measurements showed that stability increases up to DTm=45 8C for the largest oligocation moiety. In order to avoid potential toxicity of metabolites, the cationic synthon was based on the largest naturally occurring polyamine, namely spermine. Spermine is present at millimolar concentration in cells, and end-alkylation seems harmless. Amine protection followed by a,w-bishydroxylalkylation led to a diol compatible with oligonucleotide synthesis (Scheme 2). Classical dimethoxytrityl (DMT)/phosphoramidite elongation chemistry was implemented together with base-labile trifluoroacetyl (TFA) protecting groups. The spermine synthon 1 was obtained in eight steps with an overall yield of 12%. A series of decamer oligonucleotides of identical sequences N10= CACCGTAGCG appended with increasing numbers of spermine residues S was synthesized on the micromolar scale on an Expedite 8909 oligonucleotide synthetizer. Individual coupling yields were determined by spectrophotometric determination of the total amount of DMT released during each step. For the nucleoside phosphoramidites, typical coupling yields over 97% were obtained under standard conditions, whereas coupling of 1 had to be optimized to finally reach 90– 96% routinely. The synthesis was stopped with the last DMT group remaining on the oligomer to facilitate separation from truncated sequences. Oligomer cleavage from the solid support and full deprotection were performed under standard aqueous ammonia conditions. Oligomers were purified by reversed-phase HPLC or by using a Poly-Pak II cartridge. DMT was removed by using either standard acetic acid conditions or 2% aqueous TFA to give 80–250 nmol decaoligonucleotide– spermine conjugates N10Sn with up to six spermines [a] B. Pons, Dr. M. Kotera, Dr. G. Zuber, Dr. J.-P. Behr Laboratoire de Chimie G!n!tique associ! au C.N.R.S. Universit! Louis Pasteur de Strasbourg, Facult! de Pharmacie B. P. 24, 67401 Illkirch (France) Fax: (+33)390-244-306 E-mail : kotera@bioorga.u-strasbg.fr behr@bioorga.u-strasbg.fr Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author. Scheme 1. Synthesis of oligonucleotide–oligospermine conjugates NmSn.