Conjugation of Doxorubicin to siRNA Through Disulfide-based Self-immolative Linkers.

Co-delivery systems of siRNA and chemotherapeutic drugs have been developed as an attractive strategy to optimize the efficacy of chemotherapy towards cancer cells with multidrug resistance. In these typical systems, siRNAs are usually associated to drugs within a carrier but without covalent interactions with the risk of a premature release and degradation of the drugs inside the cells. To address this issue, we propose a covalent approach to co-deliver a siRNA-drug conjugate with a redox-responsive self-immolative linker prone to intracellular glutathione-mediated disulfide cleavage. Herein, we report the use of two disulfide bonds connected by a pentane spacer or a p-xylene spacer as self-immolative linker between the primary amine of the anticancer drug doxorubicin (Dox) and the 2'-position of one or two ribonucleotides in RNA. Five Dox-RNA conjugates were successfully synthesized using two successive thiol-disulfide exchange reactions. The Dox-RNA conjugates were annealed with their complementary strands and the duplexes were shown to form an A-helix sufficiently stable under physiological conditions. The enzymatic stability of Dox-siRNAs in human serum was enhanced compared to the unmodified siRNA, especially when two Dox are attached to siRNA. The release of native Dox and RNA from the bioconjugate was demonstrated under reducing conditions suggesting efficient linker disintegration. These results demonstrate the feasibility of making siRNA-drug conjugates via disulfide-based self-immolative linkers for potential therapeutic applications.

Gymnotic delivery and gene silencing activity of reduction-responsive siRNAs bearing lipophilic disulfide-containing modifications at 2'-position.

Modified oligoribonucleotides used as siRNAs bearing biolabile disulfide-containing groups at some 2'-positions were synthesized following a post-synthesis transformation of solid-supported 2'-O-acetylthiomethyl RNA, previously described. Thus, the reduction-responsive and lipophilic benzyldithiomethyl (BnSSM) modification was introduced at different locations into siRNAs targeting the Ewing sarcoma EWS-Fli1 protein. Thermal stability, serum stability and response to glutathione treatment of modified siRNAs were thoroughly investigated. Among 17 modified siRNAs, significant gene silencing activities were demonstrated for the 8 most stable siRNAs in serum (half-life > 1 h) when using a transfection reagent. Of special interest, two naked 2'-O-BnSSM siRNAs transfection exhibited a remarkable gene silencing activity after 24 h incubation. These inhibitions are consistent with an efficient gymnotic delivery demonstrated by the presence of the corresponding fluorescent siRNAs within cells.

Lipophilic 2'-O-Acetal Ester RNAs: Synthesis, Thermal Duplex Stability, Nuclease Resistance, Cellular Uptake, and siRNA Activity after Spontaneous Naked Delivery.

The in vivo application of siRNA depends on its cellular uptake and intracellular release, and this is an unsatisfactorily resolved technical hurdle in medicinal applications. Promising concepts directed towards providing efficient cellular and intracellular delivery include lipophilic chemical modification of siRNA. Here we describe chemistry for the production of modified siRNAs designed to display improved transmembrane transport into human cells while preserving the potency of the RNAi-based inhibitors. We report the synthesis and the biochemical and biophysical characteristics of 2'-O-phenylisobutyryloxymethyl (PiBuOM)-modified siRNAs and their impact on biological activity. In the case of spontaneous cellular uptake of naked PiBuOM-modified siRNA, we observed increased target suppression in human cells relative to unmodified or pivaloyloxymethyl (PivOM)-modified siRNA. We provide evidence of improved spontaneous cellular uptake of naked PiBuOM-modified siRNA and of substantial target suppression in human cells in serum-containing medium.

Synthesis, binding, nuclease resistance and cellular uptake properties of 2'-O-acetalester-modified oligonucleotides containing cationic groups.

We report on the synthesis and properties of oligonucleotides (ONs) with 2'-O-acetalester modifications containing cationic side chains in a prodrug-like approach. In the aim to improve cell penetration and nuclease resistance, various different amino- or guanidino-acetalester were grafted to 2'-OH of uridine and the corresponding phosphoramidites were incorporated into ONs. Introduction of 2'-O-(2-aminomethyl-2-ethyl)butyryloxymethyl (AMEBuOM) modification into 2'-OMe ONs leads to high resistance towards enzymatic degradation and to destabilization of duplexes with complementary RNA strand. Spontaneous uptake experiments of a twelve-mer containing ten 2'-O-AMEBuOM-U units into A673 cells showed moderate internalization of ON within the cells whereas substantial internalization of the corresponding lipophilic 2'-O-pivaloyloxymethyl ON was observed for the first time.

Nanodiamond as a vector for siRNA delivery to Ewing sarcoma cells.

The ability of diamond nanoparticles (nanodiamonds, NDs) to deliver small interfering RNA (siRNA) into Ewing sarcoma cells is investigated with a view to the possibility of in-vivo anticancer nucleic-acid drug delivery. siRNA is adsorbed onto NDs that are coated with cationic polymer. Cell uptake of NDs is demonstrated by taking advantage of the NDs' intrinsic fluorescence from embedded color-center defects. Cell toxicity of these coated NDs is shown to be low. Consistent with the internalization efficacy, a specific inhibition of EWS/Fli-1 gene expression is shown at the mRNA and protein level by the ND-vectorized siRNA in a serum-containing medium.

Harnessing subcellular-resolved organ distribution of cationic copolymer-functionalized fluorescent nanodiamonds for optimal delivery of active siRNA to a xenografted tumor in mice.

Diamond nanoparticles (nanodiamonds) can transport active drugs in cultured cells as well as in vivo. However, in the latter case, methods allowing the determination of their bioavailability accurately are still lacking. A nanodiamond can be made fluorescent with a perfectly stable emission and a lifetime ten times longer than that of tissue autofluorescence. Taking advantage of these properties, we present an automated quantification method of fluorescent nanodiamonds (FND) in histological sections of mouse organs and tumors, after systemic injection. We use a home-made time-delayed fluorescence microscope comprising a custom pulsed laser source synchronized on the master clock of a gated intensified array detector. This setup allows ultra-high-resolution images (120 Mpixels in size) of whole mouse organ sections to be obtained, with subcellular resolution and single-particle sensitivity. As a proof-of-principle experiment, we quantified the biodistribution and aggregation state of new cationic FNDs capable of transporting small interfering RNA inhibiting the oncogene responsible for Ewing sarcoma. Image analysis showed a low yield of nanodiamonds in the tumor after intravenous injection. Thus, for the in vivo efficacy assay, we injected the nanomedicine into the tumor. We achieved a 28-fold inhibition of the oncogene. This method can readily be applied to other nanoemitters with ≈100 ns lifetime.

New core-shell nanoparticules for the intravenous delivery of siRNA to experimental thyroid papillary carcinoma.

PURPOSE: Development of efficient in vivo delivery nanodevices remains a major challenge to achieve clinical application of siRNA. The present study refers to the conception of core-shell nanoparticles aiming to make possible intravenous administration of chemically unmodified siRNA oriented towards the junction oncogene of the papillary thyroid carcinoma. METHODS: Nanoparticles were prepared by redox radical emulsion polymerization of isobutylcyanoacrylate and isohexylcyanoacrylate with chitosan. The loading of the nanoparticles with siRNA was achieved by adsorption. The biological activity of the siRNA-loaded nanoparticles was assessed on mice bearing a papillary thyroid carcinoma after intratumoral and intravenous administration. RESULTS: Chitosan-coated nanoparticles with a diameter of 60 nm were obtained by adding 3% pluronic in the preparation medium. siRNA were associated with the nanoparticles by surface adsorption. In vivo, the antisense siRNA associated with the nanoparticles lead to a strong antitumoral activity. The tumor growth was almost stopped after intravenous injection of the antisense siRNA-loaded nanoparticles, while in all control experiments, the tumor size was increased by at least 10 times. CONCLUSION: This work showed that poly(alkylcyanoacrylate) nanoparticles coated with chitosan are suitable carriers to achieve in vivo delivery of active siRNA to tumor including after systemic administration.

siRNA nanoformulation against the ret/PTC1 junction oncogene is efficient in an in vivo model of papillary thyroid carcinoma.

Delivery is a very important concern for therapeutic applications of siRNA. In this study, we have used chitosan-coated poly(isobutylcyanoacrylate) nanoparticles to deliver siRNA with a complementary sequence to the fusion oncogene ret/PTC1. By screening the mRNA junction we have selected a potent siRNA sequence able to inhibit this oncogene in a model of Papillary Thyroid Carcinoma cells. This siRNA sequence has then been validated by a shRNA approach using the same sequence. Furthermore, the high ret/PTC1 inhibition has triggered a phenotypic reversion of the transformed cells. We have designed well-defined chitosan decorated nanoparticles and succeeded to reduce their size. They have allowed to protect ret/PTC1 siRNA from in vivo degradation and leading to significant tumour growth inhibition after intratumoral administration.

Delivery of siRNA to Ewing Sarcoma Tumor Xenografted on Mice, Using Hydrogenated Detonation Nanodiamonds: Treatment Efficacy and Tissue Distribution.

Nanodiamonds of detonation origin are promising delivery agents of anti-cancer therapeutic compounds in a whole organism like mouse, owing to their versatile surface chemistry and ultra-small 5 nm average primary size compatible with natural elimination routes. However, to date, little is known about tissue distribution, elimination pathways and efficacy of nanodiamonds-based therapy in mice. In this report, we studied the capacity of cationic hydrogenated detonation nanodiamonds to carry active small interfering RNA (siRNA) in a mice model of Ewing sarcoma, a bone cancer of young adults due in the vast majority to the EWS-FLI1 junction oncogene. Replacing hydrogen gas by its radioactive analog tritium gas led to the formation of labeled nanodiamonds and allowed us to investigate their distribution throughout mouse organs and their excretion in urine and feces. We also demonstrated that siRNA directed against EWS-FLI1 inhibited this oncogene expression in tumor xenografted on mice. This work is a significant step to establish cationic hydrogenated detonation nanodiamond as an effective agent for in vivo delivery of active siRNA.

Synthesis and studies on cell-penetrating peptides.

The ability of different synthetic cell penetrating peptides, as Antennapedia (wild and Phe(6) mutated penetratins), flock house virus, and integrin peptides to form complexes with a 25mer antisense oligonucleotide was compared and their conformation was determined by circular dichroism spectroscopy. The efficiency for oligonucleotide delivery into cells was measured using peptides labeled with a coumarin derivative showing blue fluorescence and the fluorescein-labeled antisense oligonucleotide showing green fluorescence. Fluorescence due to the excitation energy transfer confirmed the interaction of the antisense oligonucleotide and cell-penetrating peptides. The most efficient oligonucleotide delivery was found for penetratins. Comparison of the two types of penetratins shows that the wild-type penetratin proved to be more efficient than mutated penetratin. The paper also emphasizes that the attachment of a fluorescent label may have an effect on the conformation and flexibility of cell-penetrating peptides that must be taken into consideration when evaluating biological experiments.

Delivery of small interfering RNA. A review and an example of application to a junction oncogene.

RNA interference strategies using small interfering RNA is one of the most important discoveries in biology in recent years. This technology alongside antisense oligonucleotides is very promising and our group has focused its work on the targeting of junction oncogenes with these molecules. We have taken, as first example, papillary thyroid carcinoma. But there is a great need in delivery methods for these molecules in the treatment of cancers. Indeed, many studies have shown that small interfering RNA and antisense oligonucleotides are made efficient by various innovative delivery methods and, under these conditions, offer a powerful new therapeutic tool in cancer treatment.

Fluorescent Nanodiamond Applications for Cellular Process Sensing and Cell Tracking.

Diamond nanocrystals smaller than 100 nm (nanodiamonds) are now recognized to be highly biocompatible. They can be made fluorescent with perfect photostability by creating nitrogen-vacancy (NV) color centers in the diamond lattice. The resulting fluorescent nanodiamonds (FND) have been used since the late 2000s as fluorescent probes for short- or long-term analysis. FND can be used both at the subcellular scale and the single cell scale. Their limited sub-diffraction size allows them to track intracellular processes with high spatio-temporal resolution and high contrast from the surrounding environment. FND can also track the fate of therapeutic compounds or whole cells in the organs of an organism. This review presents examples of FND applications (1) for intra and intercellular molecular processes sensing, also introducing the different potential biosensing applications based on the optically detectable electron spin resonance of NV- centers; and (2) for tracking, firstly, FND themselves to determine their biodistribution, and secondly, using FND as cell tracking probes for diagnosis or follow-up purposes in oncology and regenerative medicine.

Cellular uptake pathways of sepiolite nanofibers and DNA transfection improvement.

Sepiolite is a nanofibrous natural silicate that can be used as a nanocarrier because it can be naturally internalized into mammalian cells, due to its nano-size dimension. Therefore, deciphering the mechanisms of sepiolite cell internalization constitutes a question interesting biotechnology, for the use of sepiolite as nanocarrier, as well as environmental and public health concerns. Though it is low, the perfectly stable and natural intrinsic fluorescence of sepiolite nanofibers allows to follow their fate into cells by specifically sensitive technics. By combining fluorescence microscopy (including confocal analysis), time-lapse video microscopy, fluorescence activated cell sorting and transmission electron microscopy, we show that sepiolite can be spontaneously internalized into mammalian cells through both non-endocytic and endocytic pathways, macropinocytosis being one of the main pathways. Interestingly, exposure of the cells to endocytosis inhibitors, such as chloroquine, two-fold increase the efficiency of sepiolite-mediated gene transfer, in addition to the 100-fold increased resulting from sepiolite sonomechanical treatment. As sepiolite is able to bind various biological molecules, this nanoparticulate silicate could be a good candidate as a nanocarrier for simultaneous vectorization of diverse biological molecules.

Antisense oligonucleotide nanocapsules efficiently inhibit EWS-Fli1 expression in a Ewing's sarcoma model.

The cytogenetic abnormality of Ewing's sarcoma is related to the presence of a balanced t(11;22) translocation expressing the EWS-Fli1 chimeric fusion protein. Oligonucleotides (ODNs) are specific compounds that inhibit gene expression at the transcriptional level. They possess a poor bioavailability and are degraded by nucleases very rapidly. Therefore, there is a strong need for the development of ODN drug delivery systems. In the present study, polyisobutylcyanoacrylate nanocapsules entrapping ODNs in their aqueous core were prepared, with high encapsulation yield (99%). Previous studies have demonstrated that such complexes were able to inhibit tumor growth in mice. Nevertheless, no information was available about their mode of action at the cellular level. The aim of this study was to investigate the efficacy of these ODN nanocapsules on cultured tumor cells. We found that nanocapsules were capable of protecting ODN against degradation. Using confocal microscopy, we observed that cell uptake and nuclear accumulation of ODNs were importantly enhanced when ODNs were associated with these nanocapsules. Consequently, a specific cellular growth inhibition and suppression of EWSFli1 fusion gene expression was noticed. In conclusion, it was demonstrated that nanocapsules as nonviral vectors show great potential for the delivery of ODNs to cells.

Formation of stable triplexes between purine RNA and pyrimidine oligodeoxyxylonucleotides.

Hybridization properties of oligodeoxyxylonucleotides (OXNs) built from pyrimidine monomers with an inverted 3'-OH group of the furanose have been studied using the gel mobility shift, UV melting and circular dichroism (CD) spectroscopy methods. Pyrimidine OXNs form triple helices with complementary purine RNA in which one OXN is parallel and another is antiparallel with respect to the RNA target. Surprisingly, no duplex formation between the pyrimidine OXNs and purine RNAs is detected. The modified triplexes are stable at pH 7. Their thermal stability depends on the number of C(G-C) triplets and, for G-rich RNA sequences, it is comparable with the stability of native DNA-RNA duplexes. The CD spectra of triplexes formed by OXNs with purine RNA targets are similar to spectra of A-type helices. A pyrimidine OXN having a clamp structure efficiently inhibits reverse transcription of murine pim-1 mRNA in vitro mediated by the Mo-MuLV reverse transcriptase.

Simultaneous cathodoluminescence and electron microscopy cytometry of cellular vesicles labeled with fluorescent nanodiamonds.

Light and Transmission Electron Microscopies (LM and TEM) hold potential in bioimaging owing to the advantages of fast imaging of multiple cells with LM and ultrastructure resolution offered by TEM. Integrated or correlated LM and TEM are the current approaches to combine the advantages of both techniques. Here we propose an alternative in which the electron beam of a scanning TEM (STEM) is used to excite concomitantly the luminescence of nanoparticle labels (a process known as cathodoluminescence, CL), and image the cell ultrastructure. This CL-STEM imaging allows obtaining luminescence spectra and imaging ultrastructure simultaneously. We present a proof of principle experiment, showing the potential of this technique in image cytometry of cell vesicular components. To label the vesicles we used fluorescent diamond nanocrystals (nanodiamonds, NDs) of size ≈150 nm coated with different cationic polymers, known to trigger different internalization pathways. Each polymer was associated with a type of ND with a different emission spectrum. With CL-STEM, for each individual vesicle, we were able to measure (i) their size with nanometric resolution, (ii) their content in different ND labels, and realize intracellular component cytometry. In contrast to the recently reported organelle flow cytometry technique that requires cell sonication, CL-STEM-based image cytometry preserves the cell integrity and provides a much higher resolution in size. Although this novel approach is still limited by a low throughput, the automatization of data acquisition and image analysis, combined with improved intracellular targeting, should facilitate applications in cell biology at the subcellular level.

Physical interactions between DNA and sepiolite nanofibers, and potential application for DNA transfer into mammalian cells.

Nanofibers of sepiolite, a natural silicate belonging to the clay minerals family, might constitute a potential promising nanocarrier for the non-viral transfer of bio-molecules. We show here that sepiolite nanofibers efficiently bind different types of DNA molecules through electrostatic interactions, hydrogen bonding, cation bridges, and van der Waals forces. Moreover, Fourier-transform infrared spectroscopy identified the external silanol groups as the main sites of interaction with the DNA. Furthermore, as a proof of concept, we show that sepiolite is able to stably transfer plasmid DNA into mammalian cells and that the efficiency can be optimized. Indeed, sonication of sepiolite 100-fold stimulated DNA transfection efficiency. These results open the way to the use of sepiolite-based biohybrids as a novel class of nanoplatform for gene transfer with potential clinical applications.

In vivo potentialities of EWS-Fli-1 targeted antisense oligonucleotides-nanospheres complexes.

The EWS/FLI-1 fusion gene, resulting from a t(11;22) translocation, plays a key role in the pathogenesis of Ewing sarcoma. Previously, we have shown that antisense oligonucleotides designed against EWS-Fli-1 inhibited tumor growth in nude mice provided they were delivered intratumorally by nanocapsules or by CTAB-coated nanospheres. In this study, we have used two types of nanospheres (designated as type 1 and type 2 nanospheres) stabilized with chitosan for both intratumoral and systemic administration of oligonucleotides. Inhibition of the tumor growth in vivo was found to be dependent on the carrier type as well as on antisense oligonucleotide modification. Indeed, whereas both types of nanospheres were efficient in reducing tumor growth after intratumoral injection, we have obtained only with type 2 nanospheres an antitumoral effect after intravenous injection in a preliminary experiment. Additionally, the anticancer efficacy of a localized modification of the EWS-Fli-1 phosphodiester/phosphorothioate chimeric antisense oligonucleotide was demonstrated. In cell culture the oligonucleotides inhibit cell growth by their antisense activity. Further investigations are needed in vivo to learn the mechanism of action of the complexes.

Short double-stranded ribonucleic acid as inhibitor of gene expression by the interference mechanism.

The rapid development of the small interfering ribonucleic acid (siRNA)-induced inhibition of the gene expression at the RNA level offers to research groups a new strategy for the understanding of gene functions. The siRNA approach is close to antisense oligonucleotide technology and takes advantage of the progress of chemically synthesized oligoribonucleotides. This approach for the mammalian cells was described by Elbashir et al. at the beginning of 2001, and in this chapter we describe methods for the design of siRNA molecules, solutions for efficiently transfecting cells, and methods for analyzing the inhibition of targeted genes. Methods for in vivo approach are also proposed.

Turning Squalene into Cationic Lipid Allows a Delivery of siRNA in Cultured Cells.

Covalent binding of squalene to siRNA has already been shown to be an interesting way of delivering siRNA in vivo. Whether squalene derivatives could also be used to deliver siRNA in cells without covalent binding similar to usual transfection with cationic lipids is the question addressed in this article. Accordingly, we investigated the activity of two squalene derivatives bearing a quaternary ammonium head group and a guanidinium group, respectively. The second derivative displayed interesting properties for delivering siRNA into cells in vitro.