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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Plasma hydrogenated cationic detonation nanodiamonds efficiently deliver to human cells in culture functional siRNA targeting the Ewing sarcoma junction oncogene.

The expression of a defective gene can lead to major cell dysfunctions among which cell proliferation and tumor formation. One promising therapeutic strategy consists in silencing the defective gene using small interfering RNA (siRNA). In previous publications we showed that diamond nanocrystals (ND) of primary size 35 nm, rendered cationic by polyethyleneimine-coating, can efficiently deliver siRNA into cell, which further block the expression of EWS/FLI-1 oncogene in a Ewing sarcoma disease model. However, a therapeutic application of such nanodiamonds requires their elimination by the organism, particularly in urine, which is impossible for 35 nm particles. Here, we report that hydrogenated cationic nanodiamonds of primary size 7 nm (ND-H) have also a high affinity for siRNA and are capable of delivering them in cells. With siRNA/ND-H complexes, we measured a high inhibition efficacy of EWS/FLI-1 gene expression in Ewing sarcoma cell line. Electron microscopy investigations showed ND-H in endocytosis compartments, and especially in macropinosomes from which they can escape before siRNA degradation occurred. In addition, the association of EWS/FLI-1 silencing by the siRNA/ND-H complex with a vincristine treatment yielded a potentiation of the toxic effect of this chemotherapeutic drug. Therefore ND-H appears as a promising delivery agent in anti-tumoral gene therapy.

Preactivated oxazaphosphorines designed for isophosphoramide mustard delivery as bulk form or nanoassemblies: synthesis and proof of concept.

Oxazaphosphorines are alkylating agents used in routine clinical practices for treatment of cancer for many years. They are antitumor prodrugs that require cytochrome P450 bioactivation leading to 4-hydroxy derivatives. In the case of ifosfamide (IFO), the bioactivation produces two toxic metabolites: acrolein, a urotoxic compound, concomitantly generated with the isophosphoramide mustard; and chloroacetaldehyde, a neurotoxic and nephrotoxic compound, arising from the oxidation of the side chains. To improve the therapeutic index of IFO, we have designed preactivated IFO derivatives with the covalent binding of several O- and S-alkyl moieties including polyisoprenoid groups at the C-4 position of the oxazaphosphorine ring to avoid cytochrome bioactivation favoring the release of the active entity and limiting the chloroacetaldehyde release. Thanks to the grafted terpene moieties, some of these new conjugates demonstrated spontaneous self-assembling properties into nanoassemblies when dispersed in water. The cytotoxic activities on a panel of human tumor cell lines of these novel oxazaphosphorines, in bulk form or as nanoassemblies, and the release of 4-hydroxy-IFO from these preactivated IFO analogues in plasma are reported.

Effects of siRNA on RET/PTC3 junction oncogene in papillary thyroid carcinoma: from molecular and cellular studies to preclinical investigations.

RET/PTC3 junction oncogene is typical of radiation-induced childhood papillary thyroid carcinoma (PTC) with a short latency period. Since, RET/PTC3 is only present in the tumour cells, thus represents an interesting target for specific therapy by small interfering RNA (siRNA). Our aim is to demonstrate in vitro and in vivo molecular and cellular effects of siRNA on RET/PTC3 knockdown for therapeutic application.First, we established a novel cell line stably expressing RET/PTC3 junction oncogene, named RP3 which was found tumorigenic in nude mice compared to NIH/3T3 mouse fibroblasts. Among four siRNAs and five concentrations tested against RET/PTC3, an efficient siRNA RET/PTC3 and an appropriate dose (50 nM) were selected which showed significant inhibition (p<0.001) of gene (RT-qPCR) and protein (Western blot) expressions. This siRNA was found efficient in RP3 cells (harbouring RET/PTC3) but non-efficient in BHP10-3 SCmice cell line (harbouring RET/PTC1) showing that a specific siRNA against fusion sequence is required to target the junction oncogene. In vitro siRNA RET/PTC3 showed significant (p<0.001) inhibitory effects on RP3 cell viability (MTT assay) and on invasion/migration (IncuCyte scratch test) with blockage of cell cycle at G0/G1 phase (flow cytometry) and induced apoptosis by caspase-3 and PARP1 cleavage (WB). After intravenous injection in nude mice, respective squalene (SQ) nanoparticles (NPs) of siRNA RET/PTC3 significantly (p<0.001) reduced RP3 tumour growth, oncogene and oncoprotein expressions, induced apoptosis and partially restored differentiation (decrease in Ki67). Hence, our findings highly support the use of siRNA RET/PTC3-SQ NPs as a new promising treatment for patients affected by PTC expressing RET/PTC3.

Influence of the internalization pathway on the efficacy of siRNA delivery by cationic fluorescent nanodiamonds in the Ewing sarcoma cell model.

Small interfering RNAs (siRNAs) are powerful tools commonly used for the specific inhibition of gene expression. However, vectorization is required to facilitate cell penetration and to prevent siRNA degradation by nucleases. We have shown that diamond nanocrystals coated with cationic polymer can be used to carry siRNAs into Ewing sarcoma cells, in which they remain traceable over long periods, due to their intrinsic stable fluorescence. We tested two cationic polymers, polyallylamine and polyethylenimine. The release of siRNA, accompanied by Ewing sarcoma EWS-Fli1 oncogene silencing, was observed only with polyethylenimine. We investigated cell penetration and found that the underlying mechanisms accounted for these differences in behavior. Using drugs selectively inhibiting particular pathways and a combination of fluorescence and electronic microscopy, we showed that siRNA gene silencing occurred only if the siRNA:cationic nanodiamond complex followed the macropinocytosis route. These results have potential implications for the design of efficient drug-delivery vectors.

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.

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.

Antitumor vectorized oligonucleotides in a model of ewing sarcoma: unexpected role of nanoparticles.

Oligonucleotides (ONs) such as antisense oligonucleotides (AS-ON) and siRNAs are used as experimental tools to study gene function and are currently being tested in clinical trials for use as therapeutic anticancer agents. However, their therapeutic use has been limited by their low physiological stability and their slow cellular uptake. The systemic delivery of sequence-specific AS-ON targeting the EWS/FLI1 gene product by a targeted, nonviral delivery system dramatically inhibits tumor growth in a murine model of Ewing's sarcoma. The nonviral delivery system uses a poly-iso-hexyl-cyanoacrylate (PIHCA)-containing polycation (chitosan) to bind and protect the AS-ON. No antitumor effect is observed using a control oligonucleotide sequence. We found here that injection of the free AS-ON stimulates tumor growth independently of its sequence and that this stimulation is abolished in the presence of nanosphere-chitosan, which exerts with the oligonucleotides a specific inhibitory effect on tumor growth. The stimulation of tumor growth is likely to be due to a polyanionic effect; indeed, a similar stimulatory response is observed upon treatment with dextran sulfate and heparin in vivo. These results suggest that ON loaded onto nanosphere-chitosan provides efficient and tumor-specific delivery, and provides protection against a polyanionic secondary effect.

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.