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Nucleic acid conjugates are promising drugs for treating gene-related diseases. Conjugating specific units like lipids, cell-penetrating peptides, polymers, antibodies, and aptamers either at the 3'- or 5'-termini of a siRNA duplex molecule has resulted in a plethora of siRNA bioconjugates with improved stabilities in bloodstream and better pharmacokinetic values than unmodified siRNAs. In this sense, lipid-siRNA conjugates have attracted a remarkable interest for their potential value in facilitating cellular uptake. In this chapter, we describe a series of protocols involving the synthesis of siRNA oligonucleotides carrying either neutral or cationic lipids at the 3'- and 5'-termini. The resulting lipid-siRNA conjugates are aimed to be used as exogenous effectors for inhibiting gene expression by RNA interference. A protocol for the formulation of lipid siRNA using sonication in the presence of serum is described yielding interesting transfection properties for cell culture without the use of transfecting agents.

Iron oxide nanoparticles (IONPs) are suitable materials for contrast enhancement in magnetic resonance imaging (MRI). Their potential clinical applications range from diagnosis to therapy and follow‐up treatments. However, a deeper understanding of the interaction between IONPs, culture media and cells is necessary for expanding the application of this technology to different types of cancer therapies. To achieve new insights of these interactions, a set of IONPs were prepared with the same inorganic core and five distinct coatings, to study their aggregation and interactions in different physiological media, as well as their cell labelling efficiency. Then, a second set of IONPs, with six different core sizes and the same coating, were used to study how the core size affects cell labelling and MRI in vitro. Here, IONPs suspended in biological media experience a partial removal of the coating and adhesion of molecules. The FBS concentration alters the labelling of all types of IONPs and hydrodynamic sizes ≥ 300 nm provide the greatest labelling using the centrifugation‐mediated internalization (CMI). The best contrast for MRI results requires a core size range between 12–14 nm coated with dimercaptosuccinic acid (DMSA) producing R2* values of 393.7 s−1 and 428.3 s−1, respectively. These findings will help to bring IONPs as negative contrast agents into clinical settings. post-print 2.788 KB

In recent years, magnetic nanoparticles have been widely investigated due to their potential in biomedical applications. For successful in vivo applications, magnetic nanoparticles must satisfy several requirements such as biocompatibility, invisibility to the immune system, high colloidal stability in biological fluids, and long blood circulation times. In this study, we have developed a formulation in which the magnetic nanoparticles are coated with bovine serum albumin to provide enhanced colloidal stability in biological fluids preserving their magnetic properties. In addition, the nanoparticles carry a chemotherapeutic drug, showing their potential as drug delivery systems. Our results reveal the influence of protein adsorption on the colloidal stability and the dynamical magnetic response of functionalized magnetic nanoparticles. Moreover, cellular internalization and in vitro cytotoxic activity in Panc-1 pancreatic cancer cells reveal enhanced cellular internalization, successful intracellular drug delivery, and efficient anticancer activity.

The synthesis of RNA molecules carrying acridine or quindoline residues at their 3′- and 5′-termini is reported. These conjugates are fully characterized by MALDI-TOF mass spectrometry. Modified siRNA duplexes carrying acridine or quindoline moieties were evaluated for inhibition of the tumor necrosis factor. The conjugates showed inhibitory properties similar to those of unmodified RNA duplexes in HeLa cells transfected with oligofectamine. The fluorescent properties of acridine derivatives allow direct observation of the cytoplasmatic distribution of modified siRNA inside the cells

8 páginas, 1 figura, 2 tablas, 4 esquemas., Novel lipid derivatives carrying amino and triazolyl groups were efficiently synthesized and covalently anchored at the 3′-termini of oligonucleotides. The desired amino-lipid conjugates were fully characterized by reversed-phase HPLC and MALDI-TOF mass spectrometry. The methodology was applied to the synthesis of lipid−siRNA designed to inhibit tumor necrosis factor (TNF-α) in order to obtain siRNAs with anti-inflammatory properties. The siRNA duplex carrying amino-lipids at the 3′-end of the passenger strand has inhibitory properties similar to those of unmodified RNA duplexes in HeLa cells, indicating that the new lipid derivatives are compatible with the RNA interference machinery., This research was supported by the Spanish Ministry of Education (Grant Nos. BFU2007- 63287 and CTQ2010-20541), the Generalitat de Catalunya (2009/SGR/208), and the Instituto de Salud Carlos III (CB06_01_0019).

Nanotechnology plays an increasingly important role in the biomedical arena. Iron oxide nanoparticles (IONPs)-labelled cells is one of the most promising approaches for a fast and reliable evaluation of grafted cells in both preclinical studies and clinical trials. Current procedures to label living cells with IONPs are based on direct incubation or physical approaches based on magnetic or electrical fields, which always display very low cellular uptake efficiencies. Here we show that centrifugation-mediated internalization (CMI) promotes a high uptake of IONPs in glioblastoma tumour cells, just in a few minutes, and via clathrin-independent endocytosis pathway. CMI results in controllable cellular uptake efficiencies at least three orders of magnitude larger than current procedures. Similar trends are found in human mesenchymal stem cells, thereby demonstrating the general feasibility of the methodology, which is easily transferable to any laboratory with great potential for the development of improved biomedical applications.

Nanomedicine nowadays offers novel solutions in cancer therapy and diagnosis by introducing multimodal treatments and imaging tools in one single formulation. Nanoparticles acting as nanocarriers change the solubility, biodistribution and efficiency of therapeutic molecules, reducing their side effects. In order to successfully apply these novel therapeutic approaches, efforts are focused on the biological functionalization of the nanoparticles to improve the selectivity towards cancer cells. In this work, we present the synthesis and characterization of novel multifunctionalized iron oxide magnetic nanoparticles (MNPs) with antiCD44 antibody and gemcitabine derivatives, and their application for the selective treatment of CD44-positive cancer cells. The lymphocyte homing receptor CD44 is overexpressed in a large variety of cancer cells, but also in cancer stem cells (CSCs) and circulating tumor cells (CTCs). Therefore, targeting CD44-overexpressing cells is a challenging and promising anticancer strategy. Firstly, we demonstrate the targeting of antiCD44 functionalized MNPs to different CD44-positive cancer cell lines using a CD44-negative non-tumorigenic cell line as a control, and verify the specificity by ultrastructural characterization and downregulation of CD44 expression. Finally, we show the selective drug delivery potential of the MNPs by the killing of CD44-positive cancer cells using a CD44-negative non-tumorigenic cell line as a control. In conclusion, the proposed multifunctionalized MNPs represent an excellent biocompatible nanoplatform for selective CD44-positive cancer therapy in vitro.

Nanotechnology plays an increasingly important role in the biomedical arena. Iron oxide nanoparticles (IONPs)-labelled cells is one of the most promising approaches for a fast and reliable evaluation of grafted cells in both preclinical studies and clinical trials. Current procedures to label living cells with IONPs are based on direct incubation or physical approaches based on magnetic or electrical fields, which always display very low cellular uptake efficiencies. Here we show that centrifugation-mediated internalization (CMI) promotes a high uptake of IONPs in glioblastoma tumour cells, just in a few minutes and via clathrin-independent endocytosis pathway. CMI results in controllable cellular uptake efficiencies at least three orders of magnitude larger than current procedures. Similar trends are found in human mesenchymal stem cells, thereby demonstrating the general feasibility of the methodology, which is easily transferable to any laboratory with great potential for the development of improved biomedical applications.

Tumor necrosis factor (TNFα) is a proinflammatory cytokine involved in the pathogenesis of inflammatory bowel disease (IBD). Although TNFα has been extensively targeted using systemic drugs, the use of antisense and small interfering RNA (siRNA) to drive down its expression at the site of inflammation should provide important advantages. In this study, native and chemically modified siRNA against TNFα was developed and characterized using a murine model of IBD. siRNA with 2′-O-methyl and propanediol modifications (siTNF-OMe-P) were resistant to nuclease degradation and provided better silencing efficacy in vitro as compared to unmodified siRNA. Every modification reduced nonspecific Toll-like receptor (TLR)-mediated immunomodulation in human peripheral blood mononuclear cells (PBMC) cells. Intrarectal administration of siTNF-OMe-P significantly ameliorated the clinical endpoints and histopathological severity in 5% dextran sulphate sodium (DSS)-treated mice as compared to unmodified and other chemically modified siRNAs. Differential gene expression assessed in siTNF-OMe-P-treated animals correlated with improved colon integrity and reduced TLR activation as compared to all treatment groups. All in all, this study demonstrates that propanediol and 2′-O-methyl modifications have profound functional consequences for siRNA efficacy in vivo. Consequently, this strategy has potential implications for therapeutic intervention in IBD and other diseases.

North bicyclo methanocarba thymidine (TN) nucleosides were substituted into siRNAs to investigate the effect of bicyclo[3.1.0]hexane 2′-deoxy-pseudosugars on RNA interference activity. Here we provide evidence that these modified siRNAs are compatible with the intracellular RNAi machinery. We studied the effect of the TN modification in a screen involving residue-specific changes in an siRNA targeting Renilla luciferase and we applied the most effective pattern of modification to the knockdown of murine tumor necrosis factor (TNF-α). We also showed that incorporation of TN units into siRNA duplexes increased their thermal stabilities, substantially enhanced serum stabilities, and decreased innate immunostimulation. Comparative RNAi studies involving the TN substitution and locked nucleic acids (LNAs) showed that the gene-silencing activities of TN-modified siRNAs were comparable to those obtained with the LNA modification. An advantage of the North 2′-deoxy-methanocarba modification is that it may be explored further in the future by changing the 2′-position. The results from these studies suggest that this modification might be valuable for the development of siRNAs for therapeutic applications., This research was supported by the European Union (MULTIFUN consortium), the Spanish Ministry of Education (BFU2007-63287, CTQ2010-20541), and the Generalitat the Catalunya (2009/SGR/208). This work was funded in part by the Center for Cancer Research, National Cancer Institute, NIH. M.T. acknowledges the JAE-Doc 2008 contract (CSIC, Spain) and the Juan de la Cierva contract (MICINN, Spain) for financial support.

The synthesis of RNA molecules carrying lipids at their 3’-termini and 5’-termini is reported. These conjugates were fully characterized by MALDI-TOF mass spectrometry and HPLC chromatography. The ability of these conjugates to silence gene expression was evaluated in the inhibition of the tumor necrosis factor. All the lipid siRNA derivatives were compatible with RNA interference machinery if transfected with oligofectamine. In the absence of a transfection agent, some lipid siRNA derivatives can exert a slight reduction of gene expression.

Oligoribonucleotide conjugates carrying nuclear localization peptide sequences at the 3?-end were prepared stepwise on a single support. The siRNA duplex carrying the nuclear localization peptide sequence at the 3?-end of the passenger strand has similar inhibitory properties as those of unmodified or cholesterol-modified RNA duplexes.

The stability of oligodeoxynucleotides to trifluoroacetic acid is studied. Pyrimidine oligonucleotides were stable in the conditions used for the removal of t-butyl groups. Oligonucleotide-3′-peptide conjugates carrying pyrimidine oligonucleotides are prepared stepwise using peptide-supports and Fmoc, t-butyl strategy. Using this strategy we have prepared an oligonucleotide-peptide conjugate containing as peptide the leucine-rich fragment of FOS, a transcription factor involved in many important cellular processes. This conjugate has a long peptide sequence with a large number of trifunctional amino acids., Project financed by Spanish Ministry of Education SAF2004-01044, BFU2005-23719- E, NAN2004-09415-C05-03 and BFU2004-02048. C. P. thanks the Spanish Ministry for a predoctoral fellowship.

(Chemical Equation Presented) 5′-Peptide oligonucleotide conjugates were prepared stepwise on a single support using Nα-Fmoc- protected amino acids and unprotected phosphate groups. The method uses commercially available reagents and is successful with most natural amino acids. The simplicity of the method may encourage researchers to prepare new oligonucleotide-peptide conjugates with novel properties., This study was supported by CICYT (BFU-2004-02048), Generalitat de Catalunya (2001/SGR/0049) and Fundació La Caixa (BM04-52-0). We thank Dr. Maria Macías, Ximena Ramírez, and Dr. Beatriz G. de la Torre for their contribution.

Iron oxide nanoparticles (IONPs) occupy a privileged position among magnetic nanomaterials with potential applications in medicine and biology. They have been widely used in preclinical experiments for imaging contrast enhancement, magnetic resonance, immunoassays, cell tracking, tissue repair, magnetic hyperthermia and drug delivery. Despite these promising results, their successful translation into a clinical setting is strongly dependent upon their physicochemical properties, toxicity and functionalization possibilities. Currently, IONPs-based medical applications are limited to the use of non-functionalized IONPs smaller than 100 nm, with overall narrow particle size distribution, so that the particles have uniform physical and chemical properties. However, the main entry of IONPs into the scene of medical application will surely arise from their functionalization possibilities that will provide them with the capacity to target specific cells within the body, and hence to play a role in the development of specific therapies. In this review, we offer an overview of their basic physicochemical design parameters, giving an account of the progress made in their functionalization and current clinical applications. We place special emphasis on past and present clinical trials.

7 páginas, 6 figuras, 1 tabla., Branched RNAs with two and four strands were synthesized. These structures were used to obtain branched siRNA. The branched siRNA duplexes had similar inhibitory capacity as those of unmodified siRNA duplexes, as deduced from gene silencing experiments of the TNF-α protein. Branched RNAs are considered novel structures for siRNA technology, and they provide an innovative tool for specific gene inhibition. As the method described here is compatible with most RNA modifications described to date, these compounds may be further functionalized to obtain more potent siRNA derivatives and can be attached to suitable delivery systems., This paper was supported by the Spanish Ministry of Education (Grants BFU2007-63287, CTQ2010-20541) and the Generalitat de Catalunya (2009/SGR/208). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008– 2011, Iniciativa Ingenio 2010, Consolider Program, and CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.