Stéphanie Foillard

SiRNA delivery with functionalized carbon nanotubes.

Carbon nanotubes (CNTs) have been studied for drug, antigen and nucleic acid delivery both in vitro and in vivo. Due to their nano-needle structure, they are supposed to cross the plasma membrane and enter directly into the cytoplasm likely upon an endocytosis-independent mechanism without inducing cell death. In this study, two cationically functionalized CNTs (CNT-PEI and CNT-pyridinium) were investigated for siRNA delivery. Both functionalized CNTs complexed siRNA and showed 10-30% silencing activity and a cytotoxicity of 10-60%. However, in terms of reduced toxicity or increased silencing activity, CNT-PEI and CNT-pyridinium did not show an added value over PEI and other standard transfection systems. Probably, the type of functionalization of carbon nanotubes might be a key parameter to obtain an efficient and non-cytotoxic CNT-based delivery system. Nevertheless, in view of the present results and importantly also of the non-degradability of CNTs, preference should currently be given to designing biodegradable carriers which mimic the needle structure of CNTs.

Synthesis and biological characterisation of targeted pro-apoptotic peptide.

We report herein the synthesis and in vitro assay of new, multimeric RGD‐peptide conjugates for cell‐targeted drug delivery. We generated a peptide scaffold comprising two functional domains, one a tumour blood vessel “homing” motif and the other a programmed cell‐death‐inducing peptide sequence. RGD peptides were selected to direct the molecular conjugate to αVβ3 integrin‐containing tumour cells. The pro‐apoptotic (Lys‐Leu‐Ala‐Lys‐Leu‐Ala‐Lys)2 peptide was found to be nontoxic outside cells, but toxic when internalized into targeted cells as it disrupted the mitochondrial membrane. The synthesis of these targeted pro‐apoptotic conjugates was carried out by assembling three different units (that is, scaffold, RGD units and pro‐apoptotic peptide) through chemoselective ligations. We show that one compound displays significant biological effect in αVβ3 integrin‐containing tumour cells.

Targeted delivery of a proapoptotic peptide to tumors in vivo.

RGD peptides recognize the αvβ3 integrin, a receptor that is overexpressed on the surface of both tumor blood vessels and cancerous cells. These peptides are powerful tools that act as single antiangiogenic molecules, but recently also have been used for tumor imaging and drug targeting. We designed the molecule RAFT-(c[-RGDfK-])4, a constrained and chemically defined entity that can be produced at clinical-grade quality. This scaffold was covalently coupled via a labile bridge to the proapoptotic peptide (KLAKLAK)2 (RAFT-RGD-KLA). A fluorescent, activatable probe was also introduced, allowing intracellular localization. At 2.5 µM, this molecule induced the intracellular release of an active KLA peptide, which in turn caused mitochondrial depolarization and cell death in vitro in tumor cells. In a mouse model, the RAFT-RGD-KLA peptide was found to prevent the growth of remote subcutaneous tumors. This study demonstrated that the antitumor peptide is capable of killing tumor cells in an RGD-dependent manner, thus lowering the nonspecific cytotoxic effects expected to occur when using cationic cytotoxic peptides. Thus, this chemistry is suitable for the design of complex, multifunctional molecules that can be used for both imaging and therapeutics, representing the next generation of perfectly controlled, targeted drug-delivery systems.

Drug development in oncology assisted by noninvasive optical imaging

Early and accurate detection of tumors, like the development of targeted treatments, is a major field of research in oncology. The generation of specific vectors, capable of transporting a drug or a contrast agent to the primary tumor site as well as to the remote (micro-) metastasis would be an asset for early diagnosis and cancer therapy. Our goal was to develop new treatments based on the use of tumor-targeted delivery of large biomolecules (DNA, siRNA, peptides, or nanoparticles), able to induce apoptosis while dodging the specific mechanisms developed by tumor cells to resist this programmed cell death. Nonetheless, the insufficient effectiveness of the vectorization systems is still a crucial issue. In this context, we generated new targeting vectors for drug and biomolecules delivery and developed several optical imaging systems for the follow-up and evaluation of these vectorization systems in live mice. Based on our recent work, we present a brief overview of how noninvasive optical imaging in small animals can accelerate the development of targeted therapeutics in oncology.

Highly efficient cell adhesion on beads functionalized with clustered peptide ligands.

Resin beads were functionalized with either clustered peptide ligands or individual peptide ligands at various ligand densities and then the beads were evaluated in a cell binding assay.

Comparative assessment of the in vitro toxicity of some functionalized carbon nanotubes and fullerenes

Functionalized fullerenes and carbon nanotubes were investigated as regards in vitro cytotoxicity and hemolytic properties. Pristine carbon nanotubes (CNTs) were first shortened to make them compatible with cellular dimensions before functional groups were appended to their surface either covalently (e.g. amine, alcohol, carboxylate) or non-covalently (adsorption and polymerization of different polyethylene glycol-based amphiphiles). C60 fullerenes were covalently functionalized with polyethylene glycols of various sizes. Cell viability was measured 24 h after exposure to the nanomaterials using MTT and LDH assays which were adapted to avoid nanomaterial interference. In vitro analysis of hemolytic properties was also performed to assess acute damage to red blood cells. While all the tested nanomaterials were found to reduce, to some extent, the cellular metabolic activity, two only affected the plasma membrane integrity, and none induced hemolysis.

Carbon nanotube-mediated delivery of budesonide to macrophages

Carbon nanotubes were functionalized with budesonide, a potent anti-inflammatory drug, and investigated for interactions with macrophages. Pristine carbon nanotubes were first shortened to make them compatible with cellular dimensions before budesonide was non-covalently appended to their surface via a specifically designed amphiphilic molecule. The resulting nanohybrids were evaluated in regards to their internalisation into macrophages and ability to inhibit pro-inflammatory cytokine secretion. In vitro experiments showed that the budesonide delivery system is readily captured by phagocytic cells and has a deep anti-inflammatory effect as it strongly inhibits IL-6 production.