Search

Supplementary Fig. S1 from Suppression of cervical carcinoma cell growth by intracytoplasmic codelivery of anti-oncoprotein E6 antibody and small interfering RNA

Supplementary Table from 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]

The advances in the microscopy technology have prompted efforts to improve the reagents required to recognize specific molecules within the intracellular environment. For high-resolution electron microscopy, conjugation of selective binders originating from the immune response arsenal to gold nanoparticles (AuNPs) as contrasting agents is the method of choice to obtain labeling tools. However, conjugation of the minimal sized 15 kDa nanobody (Nb) to AuNP remains challenging in comparison to the conjugation of 150 kDa IgG to AuNP. In here, effective Nb-AuNP assemblies are built using the selective and almost irreversible non-covalent associations between two peptide sequences deriving from a p53 heterotetramer domain variant. The 15 kDa GFP-binding Nb is fused to one dimerizing motif to obtain a recombinant Nb dimer with improved avidity for GFP while the other complementing dimerizing motif is equipped with thiols and grafted to a 2.4 nm substituted thiobenzoate-coordinated AuNP via thiolate exchange. After pegylation, the modified AuNPs are able to non- covalently anchor Nb dimers and the subsequent complexes demonstrate the ability to immunogold label GFP-protein fusions within various subcellular locations. These tools open an avenue for precise localization of targets at high resolution by electron microscopy.

Monitoring the assembly of macromolecules to design entities with novel properties can be achieved either chemically creating covalent bonds or by noncovalent connections using appropriate structural motifs. In this report, two self-associating peptides (named K3 and E3) that originate from p53 tetramerization domain were developed as tools for highly specific and noncovalent heterotetramerization of two biomolecules. The pairing/coupling preferences of K3 and E3 were first evaluated by molecular modeling data and confirmed using circular dichroism spectroscopy, size-exclusion chromatography, and biological assays. Regardless of the moieties fused to K3 and E3, these two peptides self-assembled into dimers of dimers to form bivalent heterotetrameric complexes that proved to be extremely stable inside living cells. The benefits of the multivalency in terms of avidity, specificity, and expanded functional activity were strikingly revealed when the proliferating cell nuclear antigen (PCNA), which is essential for DNA replication, was targeted using a heterotetramer presenting both an antibody fragment against PCNA and a specific PCNA binder peptide. In vitro heterotetramerization of these two known PCNA ligands increased their binding efficiencies to PCNA up to 80-fold compared to the best homotetramer counterpart. In cellulo, the heterotetramers were able to efficiently inhibit DNA replication and to trigger cell death. Altogether, we demonstrate that these two biselective self-assembling peptidic domains offer a versatile noncovalent conjugation method that can be easily implemented for protein engineering. papers2://publication/uuid/89537C97-09B4-4D8F-B955-FFAF0CF7E08A

Simple Summary The focal adhesion kinase (FAK) is over-expressed in a variety of human tumors and is involved in many aspects of the metastatic process. This has led to the development of small inhibitors of FAK kinase function which are currently evaluated in clinical trials. We demonstrate here that this class of inhibitors, while decreasing melanoma cell migration, increases invadopodia activity in metastatic melanoma cells. Searching for an alternative strategy to inhibit the oncogenic activity of FAK, we show that inhibiting FAK scaffolding function using a small peptide altering FAK–paxillin interactions reduces both migration and invadopodia-mediated matrix degradation in metastatic melanoma cells. Abstract The nonreceptor tyrosine kinase FAK is a promising target for solid tumor treatment because it promotes invasion, tumor progression, and drug resistance when overexpressed. Investigating the role of FAK in human melanoma cells, we found that both in situ and metastatic melanoma cells strongly express FAK, where it controls tumor cells’ invasiveness by regulating focal adhesion-mediated cell motility. Inhibiting FAK in human metastatic melanoma cells with either siRNA or a small inhibitor targeting the kinase domain impaired migration but led to increased invadopodia formation and extracellular matrix degradation. Using FAK mutated at Y397, we found that this unexpected increase in invadopodia activity is due to the lack of phosphorylation at this residue. To preserve FAK–Src interaction while inhibiting pro-migratory functions of FAK, we found that altering FAK–paxillin interaction, with either FAK mutation in the focal adhesion targeting (FAT) domain or a competitive inhibitor peptide mimicking paxillin LD domains drastically reduces cell migration and matrix degradation by preserving FAK activity in the cytoplasm. In conclusion, our data show that targeting FAK–paxillin interactions could be a potential therapeutic strategy to prevent metastasis formation, and molecules targeting this interface could be alternative to inhibitors of FAK kinase activity which display unexpected effects.

Immunotoxins are emerging candidates for cancer therapeutics. These biomolecules consist of a cell-targeting protein combined to a polypeptide toxin. Associations of both entities can be achieved either chemically by covalent bonds or genetically creating fusion proteins. However, chemical agents can affect the activity and/or stability of the conjugate proteins, and additional purification steps are often required to isolate the final conjugate from unwanted byproducts. As for fusion proteins, they often suffer from low solubility and yield. In this report, we describe a straightforward conjugation process to generate an immunotoxin using coassociating peptides (named K3 and E3), originating from the tetramerization domain of p53. To that end, a nanobody targeting the human epidermal growth factor receptor 2 (nano-HER2) and a protein toxin fragment from Pseudomonas aeruginosa exotoxin A (TOX) were genetically fused to the E3 and K3 peptides. Entities were produced separately in Escherichia coli in soluble forms and at high yields. The nano-HER2 fused to the E3 or K3 helixes (nano-HER2-E3 and nano-HER2-K3) and the coassembled immunotoxins (nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX) presented binding specificity on HER2-overexpressing cells with relative binding constants in the low nanomolar to picomolar range. Both toxin modules (E3-TOX and K3-TOX) and the combined immunotoxins exhibited similar cytotoxicity levels compared to the toxin alone (TOX). Finally, nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX evaluated on various breast cancer cells were highly potent and specific to killing HER2-overexpressing breast cancer cells with IC50 values in the picomolar range. Altogether, we demonstrate that this noncovalent conjugation method using two coassembling peptides can be easily implemented for the modular engineering of immunotoxins targeting different types of cancers.

Efficiency of polyethylenimine (PEI) for nucleic acid delivery is affected by the size of the carrier and length of the nucleic acids. For instance, PEIs with molecular weights between 10-30kDa provide optimal DNA delivery activity whereas PEIs with molecular weights below 1.8kDa are ineffective. The activity of PEI is also severely diminished by substitution of DNA for shorter nucleic acids such as mRNA or siRNA. Here, through chemical modification of the primary amines to aromatic domains we achieved nucleic acid delivery by the 1.8kDa polyethylenimine (PEI) particles. This modification did not affect the PEI buffering abilities but enhanced its pH-sensitive aggregation, enabling stabilization of the polyplex outside the cell while still allowing nucleic acid release following cellular entry. The aromatic PEIs were then evaluated for their gene, mRNA, siRNA and 2'O-methyl phosphorothioate oligonucleotide in vitro transfection abilities. The salicylamide-grafted PEI showed to be a reliable carrier for delivering nucleic acids with cytoplasmic activity such as the mRNA and siRNA or nuclear diffusible oligonucleotide. It was then further equipped with polyethyleneglycol (PEG) and the delivery efficiency of the copolymer was tested in vivo for regeneration of dystrophin in the muscle of mdx mouse through a 2'O-methyl phosphorothioate-mediated splicing modulation. Intramuscular administration of polyplexes resulted in dystrophin-positive fibers in a mouse model of Duchenne muscular dystrophy without apparent toxicity. These findings indicate that precise modifications of low molecular weight PEI improve its bio-responsiveness and yield delivery vehicles for nucleic acids of various types in vitro and in vivo.

International audience; Therapeutic monoclonal antibodies benefit to patients and the conjugation to gold nanoparticles (AuNPs) might bring additional activities to these macromolecules. However, the behavior of the conjugate will largely depend on the bulkiness of the AuNP and small sizes are moreover preferable for diffusion. Water-soluble thiolate-protected AuNPs having diameters of 2–3 nm can be synthesized with narrow polydispersity and can selectively react with incoming organic thiols via a SN2-like mechanism. We therefore synthesized a mixed thionitrobenzoic acid- , thioaminobenzoic acid-monolayered AuNP of 2.4 nm in diameter and developed a site-selective conjugation strategy to link the AuNP to Cetuximab, an anti-epidermal growth factor receptor (EGFR) antibody used in clinic. The water-soluble 80 kDa AuNP was fully characterized and then reacted to the hinge area of Cetuximab, which was selectively reduced using mild concentration of TCEP. The conjugation proceeded smoothly and could be analyzed by polyacrylamide gel electrophoresis, indicating the formation of a 1:1 AuNP-IgG conjugate as the main product. When added to EGFR expressing glioblastoma cells, the AuNP-Cetuximab conjugate selectively bound to the cell surface receptor, inhibited EGFR autophosphorylation and entered into endosomes like Cetuximab. Altogether, we describe a simple and robust protocol for a site-directed conjugation of a thiolate-protected AuNP to Cetuximab, which could be easily monitored, thereby allowing to assess the quality of the product formation. The conjugated 2.4 nm AuNP did not majorly affect the biological behavior of Cetuximab, but provided it with the electronic properties of the AuNP. This offers the ability to detect the tagged antibody and opens application for targeted cancer radiotherapy.

For biological and medical applications, a definable nanomaterial and knowledge on its fate after administration are highly recommended if not mandatory. Here, we synthesized a water-soluble gold nanocluster by sodium borohydride reduction of chloroauric acid in the presence of 5,5′-dithio-bis(2-nitrobenzoic acid). The resulting gold nanocluster displayed the physical characteristics of a cluster containing an inner core of about 100 gold atoms that was surrounded by an organic monolayer made of about 30 thioaminobenzoic acids (TAB) and 14 anionic thionitrobenzoic acids (TNB). The mixed TAB-,TNB-protected gold nanocluster reacted well in water with thiolated peptides containing a nuclear localization signal (NLS) or a nuclear export signal (NES) mainly by exchange of the TNB, providing gold nanoclusters equipped with 8–9 intracellular active peptides and a remaining ligand coverage consisting mostly of the zwitterion TAB. The behavior of these peptide–gold nanoclusters inside the cytosol and nucleus of cells was then assayed using an electroporation procedure allowing transient plasma membrane permeability. Light and electron microscopy observations demonstrated a consistent inflow and diffusion of the gold nanoclusters into the cytosol. Inside the living cells, the distribution of the gold nanoparticles was specifically driven by the appended signal peptides in a manner similar to the distribution of NLS and NES-bearing proteins, demonstrating diffusion ability, stability, and usage of these definable ligand-substituted gold nanoclusters for intracellular applications.

We report here the synthesis of a diblock linear polymer of oligo(benzylethylenimine)-b-polyethylenimine (OBzEI-PEI) and investigate its gene delivery properties. The linear copolymer OBzEI-PEI was prepared in a straightforward manner by acidic hydrolysis of a diblock polyoxazoline, which had been made by sequential polymerization of 4-benzyl-2-ethyl-2-oxazoline followed by 2-ethyl-2-oxazoline. pH titration and DNA complexation profiles of the new polymer are similar to regular linear PEIs, but with higher gene transfection efficiencies in various cell lines despite a decreased cellular uptake of plasmid DNA. Further experiments suggest that the OBzEI tail complements the intrinsic proton-sponge endosomolytic activities of PEI with an acid pH-sensitive membrane-perturbing activity.

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 (t1/2 = 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.

ObjectiveTo 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.MethodsEvaluation 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.ResultsWe 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.ConclusionsOur data point toward an important role for miR-20a in the regulation of pro-inflammatory cytokines release, by controlling ASK1 expression in RA FLS.

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.

Injection of hyaluronic acid (HA)-based hydrogels has proven to provide many therapeutic benefits. To increase the stability of HA-based products against enzymatic digestion, we modified hyaluronic acid by grafting various amino acids on its carboxylic group and then evaluated the enzymatic stability of the various conjugates in presence of a hyaluronidase. Our results showed that all amino acid-modified HA polymers were more resistant to degradation compared to the native HA albeit with variation according to the amino acids. Amino acids with carboxylate groups such as aspartic acid or with hydroxyl functions (threonine, serine or tyrosine) conferred a particularly strong resistance to HA towards enzymatic digestion. The HA-amino acid products were then cross-linked with butanediol diglycidyl ether (BDDE). The swelling properties of the formed hydrogels appeared close to native HA whereas the increased resistance towards hyaluronidase digestion remained. These results suggest that amino acid-modified HA derivatives can become promising material for viscosupplementation or drug delivery.

In order to increase the resistance of hyaluronic acid towards enzymatic digestion, we prepared N-alanyl-hyaluronamide derivatives using different amidation methods performed either in water, water/acetonitrile mixture or anhydrous dimethylformamide. Our results indicate that amidation of hyaluronic acid in an anhydrous solvent is effective and led to a degree of substitution of the carboxylic groups up to 100%. We also demonstrated the N-alanyl-hyaluronamides present enhanced resistance towards enzymatic digestion while forming solutions with similar viscosities than solutions of hyaluronic acids of similar lengths.

Hyaluronic acid (HA) is widely used for numerous medical applications, such as viscosupplementation, eye surgery and drug delivery. A broad range of HA-based materials have been developed and described for the enhancement, modulation and control of its therapeutic action, based on chemical modification of polysaccharides. The purpose of this paper is to review the various chemical modification methods and synthetic routes to obtain HA derivatives, encompassing all applications.

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.

The human organism and its constituting cells rely on interplay between multiple proteins exerting specific functions. Progress in molecular biotechnologies has facilitated the production of recombinant proteins. When administrated to patients, recombinant proteins can provide important healthcare benefits. To date, most therapeutic proteins must act from the extracellular environment, with their targets being secreted modulators or extracellular receptors. This is because proteins cannot passively diffuse across the plasma membrane into the cytosol. To expand the scope of action of proteins for cytosolic targets (representing more than 40% of the genome) effective methods assisting protein cytosolic entry are being developed. To date, direct protein delivery is extremely tedious and inefficient in cultured cells, even more so in animal models of pathology. Novel techniques are changing this limitation, as recently developed in vitro methods can robustly convey large amount of proteins into cell cultures. Moreover, advances in protein formulation or protein conjugates are slowly, but surely demonstrating efficiency for targeted cytosolic entry of functional protein in vivo in tumor xenograft models. In this review, various methods and recently developed techniques for protein transport into cells are summarized. They are put into perspective to address the challenges encountered during delivery.

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]

Recombinant proteins with cytosolic or nuclear activities are emerging as tools for interfering with cellular functions. Because such tools rely on vehicles for crossing the plasma membrane we developed a protein delivery system consisting in the assembly of pyridylthiourea-grafted polyethylenimine (πPEI) with affinity-purified His-tagged proteins pre-organized onto a nickel-immobilized polymeric guide. The guide was prepared by functionalization of an ornithine polymer with nitrilotriacetic acid groups and shown to bind several His-tagged proteins. Superstructures were visualized by electron and atomic force microscopy using 2 nm His-tagged gold nanoparticles as probes. The whole system efficiently carried the green fluorescent protein, single-chain antibodies or caspase 3, into the cytosol of living cells. Transduction of the protease caspase 3 induced apoptosis in two cancer cell lines, demonstrating that this new protein delivery method could be used to interfere with cellular functions.

Oligonucleotide delivery is a crucial issue for therapeutical purposes and is often addressed by conjugation to short cationic peptides although with controversial results. To further examine this mechanism, a 15-mer anionic oligonucleotide was conjugated to a cationic peptide in order to obtain a diblock compound with an overall positive charge with aggregation properties. These microaggregates were efficiently internalized in cells via the expeditious pathway used by commercial gene delivery systems. Moreover, stability of the duplex formed with the complementary sequence increased without inhibiting oligonucleotide enzyme recognition as shown by the properties of the conjugate to prime chain elongation by Taq DNA polymerase in a linear amplification/sequencing process.

Protein delivery is emerging as an interesting alternative to gene delivery. We have used our experience with transfection to develop a technique for efficient delivery of anionic proteins such as antibodies into the cytoplasm of cells. As for DNA, when complexed with cationic lipids, large amounts of proteins are shown to enter adherent cells via ubiquitously expressed syndecans. However, protein surface area rather than electric charge ratio governs the delivery characteristics. Delivery of anti-beta-actin and anti-alpha-tubulin IgG's leads to fiber depolymerization. Intracellular delivery of an antibody could thus be regarded as another method for interfering with gene activity.

Medical application of siRNAs relies on methods for delivering nucleic acids into the cytosol. Synthetic carriers, which assemble with nucleic acids into delivery systems, show promises for cancer therapy but efficiency remains to be improved. In here, the effectiveness of pyridylthiourea‐polyethylenimine (πPEI), a siRNA carrier that favors both polyplex disassembly and endosome rupture upon sensing the acidic endosomal environment, in 3 experimental models of hepatocellular cancer is tested. The πPEI‐assisted delivery of a siRNA targeting the polo‐like kinase 1 into Huh‐7 monolayer produces a 90% cell death via a demonstrated RNA interference mechanism. Incubation of polyplex with Huh‐7 spheroids leads to siRNA delivery into the superficial first cell layer and a 60% reduction in spheroid growth compared to untreated controls. Administration of polyplexes into mice bearing subcutaneous implanted Huh‐7Luc tumors results in a reduced tumor progression, similar to the one observed in the spheroid model. Altogether, these results support the in vivo use of synthetic and dedicated polymers for increasing siRNA‐mediated gene knockdown, and their clinical promise in cancer therapeutics.

Labeling nuclear proteins with electron dense probes in living cells has been a major challenge due to their inability to penetrate into nuclei. We developed a lipid-based approach for delivering antibodies coupled to 0.8 nm ultrasmall gold particles into the nucleus to label RNA polymerase II. Focussed Ion Beam slicing coupled to Scanning Electron Microscopy (FIB/SEM) enabled visualization of entire cells with probe localization accuracy in the 10 nm range.

International audience; The activity of synthetic interfering nucleic acids (siRNAs) relies on the capacity of delivery systems to efficiently transport nucleic acids into the cytosol of target cells. The pyridylthiourea-grafted 25KDa polyethylenimine (πPEI) is an excellent carrier for siRNA delivery into cells and it was extensively investigated in this report. Quantification of the siRNA-mediated gene silencing efficiency indicated that the πPEI specific delivery activity at the cell level may be measured and appears relatively constant in various cell lines. Delivery experiments assaying inhibitors of various entry pathways or concanamycin A, an inhibitor of the H+/ATPase vacuolar pump showed that the πPEI/siRNA polyplexes did not require any specific entry mode but strongly relied on vacuolar acidification for functional siRNA delivery. Next, πPEI polyplexes containing a siRNA targeting the transcription factor HIF-1α, known to be involved in tumor progression, were locally injected into mice xenografted with a human glioblastoma. A 55% reduction of the level of the target mRNA was observed at doses comparable to those used in vitro when the πPEI delivery activity was calculated per cell. Altogether, our study underscores the usefulness of "simple"/rough cationic polymers for siRNA delivery despite their intrinsic limitations. The study underscores as well as that bottom-up strategies make sense. The in vitro experiments can precede in vivo administration and be of high value for selection of the carrier with enhanced specific delivery activity and parallel other research aiming at improving synthetic delivery systems for resilience in the blood and for enhanced tissue-targeting capacity.

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.

Retrovirus Gag precursor (PrGag) proteins direct the assembly of roughly spherical immature virus particles, while after proteolytic processing events, the Gag capsid (CA) and nucleocapsid (NC) domains condense on viral RNAs to form mature retrovirus core structures. To investigate the process of retroviral morphogenesis, we examined the properties of histidine-tagged (His-tagged) Moloney murine leukemia (M-MuLV) capsid plus nucleocapsid (CANC) (His-MoCANC) proteins in vitro. The His-MoCANC proteins bound RNA, possessed nucleic acid-annealing activities, and assembled into strand, circle (or sphere), and tube forms in the presence of RNA. Image analysis of electron micrographs revealed that tubes were formed by cage-like lattices of CANC proteins surrounding at least two different types of protein-free cage holes. By virtue of a His tag association with nickel-chelating lipids, His-MoCANC proteins also assembled into planar sheets on lipid monolayers, mimicking the membrane-associated immature PrGag protein forms. Membrane-bound His-MoCANC proteins organized into two-dimensional (2D) cage-like lattices that were closely related to the tube forms, and in the presence of both nickel-chelating lipids and RNAs, 2D lattice forms appeared similar to lattices assembled in the absence of RNA. Our observations are consistent with a M-MuLV morphogenesis model in which proteolytic processing of membrane-bound Gag proteins permits CA and NC domains to rearrange from an immature spherical structure to a condensed mature form while maintaining local protein-protein contacts.

We have used an in vitro system that mimics the assembly of immature Moloney murine leukemia virus (M-MuLV) particles to examine how viral structural (Gag) proteins oligomerize at membrane interfaces. Ordered arrays of histidine-tagged Moloney capsid protein (his-MoCA) were obtained on membrane bilayers composed of phosphatidylcholine (PC) and the nickel-chelating lipid 1,2-di-O-hexadecyl-sn-glycero-3-(1′-2”-R-hydroxy-3′N-(5-amino-1-carboxypentyl)iminodiacetic acid)propyl ether (DHGN). The membrane-bound arrays were analyzed by electron microscopy (EM) and atomic force microscopy (AFM). Two-dimensional projection images obtained by EM showed that bilayer-bound his-MoCA proteins formed cages surrounding different types of protein-free cage holes with similar cage holes spaced at 81.5-Å distances and distances between dissimilar cage holes of 45.5Å. AFM images, showing topological features viewed near the membrane-proximal domain of the his-MoCA protein, revealed a cage network of only symmetrical hexamers spaced at 79-Å distances. These results are consistent with a model in which dimers constitute structural building blocks and where membrane-proximal and distal his-MoCA regions interact with different partners in membrane-bound arrays.

It has been shown previously that two structural domains of bleomycin mediate the interaction of the antitumor antibiotic with DNA. At least three lines of evidence indicate that the metal binding domain is the predominant partner, and is responsible for the sequence selectivity of DNA cleavage observed for Fe(II)*BLM. The bithiazole + C-terminus has been shown previously to bind to DNA, but not to exhibit any sequence selectivity. Presently, it is shown that the bithiazole + C- terminus may also exhibit sequence selectivity, albeit not in the same fashion as bleomycin itself. The potential of this selectivity to contribute to the recognition of high affinity sites on DNA by bleomycin is discussed.

Intracellular delivery of functionally active proteins into cells is emerging as a novel strategy for research and therapeutic applications. Here, we present the properties of a self-assembling pyridylthiourea-modified polyethylenimine (πPEI), which interacts with proteins and promotes their delivery into the cytosol of mammalian cells. In aqueous medium at pH 7.4, self-association of πPEI in the presence of green fluorescent proteins (GFP) leads to supramolecular protein-entrapped assemblies. These assemblies protect GFP from losing its fluorescence upon pH variation and assist delivery/translocation into the cytosol of mammalian cells via the endocytic pathway. The scope of application of this delivery system was extended to antibodies against intracellular targets as illustrated using a monoclonal antibody directed against the HPV-16 viral E6 oncoprotein and an antibody directed against the threonine-927 phosporylation site of the EG5 kinesin spindle protein. The πPEI-mediated delivery of native anti-E6 antibodies or anti-E6 antibodies equipped with a nuclear localization signal (NLS), led to regeneration of the p53 tumor suppression protein in E6-transformed CaSki cells. Delivery of functionally active anti-EG5 antibodies, with the same polymer, reduced HeLa cell viability and appeared to perturb, as expected, chromosome segregation during mitosis. Altogether, these results provide an easy to use delivery system for extending the scope of application of antibodies for epitope recognition within living cells and may provide novel opportunities for selective interference of cell function by a steric hindrance modality.

Biolabile cationic lipids were developed for efficient intracellular delivery of DNA and siRNA.The 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.They 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.The 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.

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.

DNA ligation is the weak link in the chain of gene cloning. We have developed a straightforward nonenzymatic alternative to this reaction that employs easily available commercial reagents. The method uses the affinity of distamycin for the minor groove to join DNA ends together. Phosphodiester bonds are formed after cyanoimidazole-promoted phosphate activation in the presence of manganese(II) cations. When transfected into eukaryotic cells, the chemically ligated plasmid is transcribed even more efficiently than after enzymatic ligation. At present this technique compares favorably with T4 ligation for AT-rich cohesive termini, but in principle it could be extended to any restriction site.

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 (πPEΙ) 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/πPEΙ 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.

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.

Prostate cancer is a leading cause of death among men in Western countries. Whereas the survival rate approaches 100% for patients with localized cancer, the results of treatment in patients with metastasized prostate cancer at diagnosis are much less successful. The patients are usually presented with a variety of treatment options, but therapeutic interventions in prostate cancer are associated with frequent adverse side effects. Gene therapy and oncolytic virus therapy may constitute new strategies. Already a wide variety of preclinical studies has demonstrated the therapeutic potential of such approaches, with oncolytic prostate-specific adenoviruses as the most prominent vector. The state of the art and future prospects of gene therapy in prostate cancer are reviewed, with a focus on adenoviral vectors. We summarize advances in adenovirus technology for prostate cancer treatment and highlight areas where further developments are necessary.

In this study, the ability of synthetic fluorinated lipospermines to bind DNA and siRNA was investigated and the transfection efficiency and toxicity of the resulting lipoplexes in cell lines were evaluated. Three lipopolyamines displaying fluorous tags close to their cationic polar head ("HFP" polyamines) were synthesized. Their ability to condense pDNA and siRNA, and to form nanoparticles were characterized. Lipoplex stability was investigated in the presence of different surface active compounds and was shown to be significantly improved due to the presence of the fluorous tags. Transfection efficiencies were studied in HepG2 and 911 cell lines, and compared to that of DOGS, DOTAP, and Lipofectamine 2000. Also, the ability of these compounds to deliver nucleic acids into cells in the presence of high concentration of serum was quantified. By incorporating two fluorous tags in the direct vicinity of the polycationic head group of the lipospermines, we show efficient pDNA and siRNA formulation, and delivery to cultured cells. Fluorinated lipoplexes exhibit improved stability in the presence of amphiphilic compounds and retain high transfection efficiency in the presence of 50-75% serum. These results demonstrate that lipospermines displaying fluorous tags close to their cationic polar head bind to and deliver pDNA and siRNA with high cell viability in different cell lines. They are efficient non-viral vectors that exhibit remarkable serum compatibility.

Although a great number of cationic lipids have been designed and evaluated as gene delivery systems, there is still a need for improvement of nonviral vectors. Recently, cationic lipids incorporating terminal fluoroalkyl segments ("FHP" lipids) have been described to display remarkable transfection potency. Here, we describe the synthesis of a new family of fluorinated triblock cationic lipids in which a fluorous segment lays between the cationic and the lipophilic parts of the molecule ("HFP" lipids). The compounds were designed so their self-assembly would offer enhanced resistance toward the host's degradation mechanisms mediated by lipophilic insertion. Self-assembly properties of these cationic lipids were evaluated at the air-water interface where they collapse in a highly ordered liquid phase. The HFP lipids efficiently condense DNA, and the resulting lipoplexes display enhanced resistance to amphiphilic agents when compared to nonfluorinated or FHP cationic lipids. Transfection properties of the fluorinated vectors, alone or as mixtures with different helper lipids (DOPE and a fluorinated analogue of DOPE), were then investigated on different cell lines (BHK-21, HepG2, and HeLa) and compared to those of the reference cationic lipid DOTAP. Data show that impermeabilization of the lipidic phase by fluorous segments alter significantly the gene transfection activities. Remarkably, incorporation of DOPE within the lipoplexes provides the particles with high gene transfection activity without reducing their resistance to amphiphilic agents.

Splice-correcting phosphorothioate RNA antisense oligonucleotides with 2'-O-methyl modifications (ASO) are promising therapeutic agents for several disorders caused by aberrant splicing. However, their usefulness is hindered by the lack of efficient delivery. Unmodified 25 kDa polyethylenimine (PEI) has shown potential for plasmid delivery but seems to be less efficient for short nucleic acid sequences. Herein, we have evaluated several amino acid modified PEI molecules as carriers for ASO. By characterization of their properties, such as size, stability and transfection into mammalian cells, we have identified tyrosine-modified PEI (PEIY) as an efficient ASO delivery system. HeLa705 cells containing an aberrant luciferase gene, interrupted by a mutated beta-globin intron, were used to assess the splice correction effectiveness mediated by the various modified PEI/ASO polyplexes. PEIY has a self-assembly nature, as opposed to the highly cationic parent polymer, which is relevant for the stability of the PEIY/ASO complexes. As a result, at an optimal ratio of 20:1 (+/-), the complexes that formed significantly corrected the splicing on both the mRNA and the protein levels. ASO formulated with PEIY enhanced luciferase activity up to 450-fold. This increase was three times higher than that produced by the commercially available transfection agent Lipofectamine. PEIY/ASO polyplexes resulted in at least 80% correct splicing of the transcript. Moreover, extremely low doses of ASO (0.025 microM) showed significant splice correction represented by 150-fold increase of luciferase activity and 47% mRNA correction. Our findings suggest key parameters for formulating active complexes and reveal a new platform that can be further developed for ASO in vivo targeting.

The potential effect of different classes of steroids on the expression of acetylcholine receptors (AChR) was studied in different primary cultures of newborn-rat skeletal muscle cells. Comparison among three techniques for preparing newborn skeletal muscle cells showed that these systems were equivalent to study AChR expression. Only corticoids stimulated myogenesis as a twofold increase in AChR expression indicated. Among the corticoids, the glucocorticoids were the more potent, whereas the mineralocorticoid aldosterone had less marked effect. The sex hormones progesterone and testosterone partially blocked these effects, without inducing any significant effect when given alone. The steroids tested differed in efficacy in correlation with their different chemical structures. Among the glucocorticoids a clear structure-activity relationship could be established. These results emphasize the specificity of corticoid action on muscle cells and suggest an explanation for the effects induced by glucocorticoids used in treating human muscular or neuromuscular diseases.

Progress in the design of gene delivery systems is of utmost importance for cancer gene therapy since several physiological and intracellular barriers remain. We previously developed a technology for condensing a single gene into a single and stable globular nanometric system. In this manuscript, we have decorated the nanometric particles with cyclic RGD ligands in order to target endothelial cells. The potential of these artificial viruses as targeted gene delivery vehicles is demonstrated in vitro with alpha(v)beta(3) integrin-expressing primary endothelial cells.

Deoxyribose nucleic acid or DNA is a linear polymer in the form of a double strand, synthesised by sequential polymerisation of a large number of units chosen from among the nucleic bases called purines (adenosine A and guanosine G) and pyrimidines (cytosine C and thymidine T). DNA contains all the genetic information required for life. It exists in the form of a limited number (a few dozen) of very big molecules, called chromosomes. This genetic information is first of all transcribed. In this process, a restricted fragment of the DNA called a gene is copied in the form of ribonucleic acid, or RNA. This RNA is itself a polymer, but with a single strand in which the sequence of nucleic acids is schematically analogous to the sequence on one of the two strands of the transcribed DNA. Finally, this RNA is translated into a protein, yet another linear polymer. The proteins make up the main part of the active constituents ensuring the survival of the cell. Any loss of information, either by mutation or by deletion of the DNA, will cause an imbalance in the cell's metabolism that may in turn lead to incurable pathologies. Several strategies have been developed to reduce the consequences of such genetic deficiencies or, more generally, to act, by amplifying or suppressing them, on the mechanisms leading from the reading of the genetic information to the production of proteins: Strategies aiming to introduce synthetic DNA or RNA, which selectively block the expression of certain genes, are now being studied by an increasing number of research scientists and pharmacologists. They use antisense oligodeoxyribonucleotides or interfering oligoribonucleotides and they already have clinical applications. This kind of therapy is often called gene pharmacology. Other, more ambitious strategies aim to repair in situ mutated or incomplete DNA within the chromosomes themselves, by introducing short sequences of DNA or RNA which recognise and take the place of mutations. This is the underlying principle of genetic correction. Yet other strategies aim to reintroduce the deficient DNA fragments into the cells in the form of genes. Indeed, in certain diseases, the only solution is to bring genetic information back into the cells by transferring exogeneous DNA into the cell nucleus. This approach goes by the name of gene therapy.