Oligonucleotide delivery: A patent review (2010-2013)

Introduction: The use of aptamers, antisense technology and RNA interference has allowed nucleic acids to be considered as promising alternatives to classical drugs. However, nucleic acids face several obstacles in the creation of effective nucleic acid drugs. The development of these approaches has strengthened the pipeline with an increasing number of these therapies in clinical trials.Areas covered: This review covers research and patent literature from the last three years, focusing on the development of safe and effective non-viral drug delivery systems for the treatment of diseases such as cancer or genetic disorders by using oligonucleotides.Expert opinion: The therapeutic applications of oligonucleotides have overcome multiple obstacles, especially in biodistribution and cellular internalization. Cationic lipids are the most used vehicles for the preparation of novel formulations. Combinatorial libraries of these compounds and the use of solid lipid nanoparticles carrying these synthetic cationic lipids (cholesterol and PEG) have enhanced cellular uptake and biocompatibility of nucleic acids. Besides this extensive use, synthesis of oligonucleotides covalently linked to lipids has also emerged as a promising alternative to formulations. The use of peptides alone or in combination with lipids is an expanding field for oligonucleotide delivery. Polymeric platforms are also good candidates as they showed improved cellular uptake, biodegradability, biocompatibility and the possibility of incorporating several components, such as ligands for receptor-mediated endocytosis and molecules, to facilitate endosomal escape. Finally, nanomaterials may also play an important role in the future. The last developments showed improvement in in vivo efficacy, thus gaining a foothold in therapeutics.This work is supported by the European Commission (Grant NMP4-LA-2011-262943, MULTIFUN), by the Spanish Ministry of Education (Grant CTQ2010-20541), the Generalitat de Catalunya (2009/SGR/208) and the Instituto de Salud Carlos III (CB06_01_0019).Peer reviewe

Synthesis and Properties of Oligodeoxynucleotides Carrying 2-Aminopurine

The use of benzoyl, isobutyryl and dimethylaminomethylidene groups for the protection of the exocyclic amino function of 2-aminopurine during oligonucleotide synthesis has been investigated. Best results in the synthesis were obtained with the monomers of 2-aminopurine protected with the isobutyryl groupThis work was supported by the Spanish Ministry of Education (grant CTQ2010-20541) and the Generalitat de Catalunya (2009/SGR/208).Peer reviewe

1-[2,3-Bis(tetradecyloxy)propyl]-3-[2-(piperazin-1-yl)ethyl]urea

Starting from 2,3-bis(tetradecyloxy)propan-1-amine (1), the synthesis of the target compound 1-[2,3-bis(tetradecyloxy)propyl]-3-[2-(piperazin-1-yl)ethyl]urea (2) is reported. The title compound was characterized by 1H-NMR, 13C-NMR and ESI/MS analysis.This work is supported by grants of the Spanish Ministry of Economy (MINECO) (CTQ2014-52588-R, RTC-2014-2038-1, CTQ2014-61758-EXP), Generalitat de Catalunya 2014/SGR/624) and the Instituto de Salud Carlos III (CB06_01_0019). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008-2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund. We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).Peer reviewe

Synthesis of oligonucleotides carrying fluorescently labelled O6-alkylguanine for measuring hAGT activity

O6-alkylguanine-DNA-alkyltransferase (hAGT) activity provides resistance to cancer chemotherapeutic agents and its inhibition enhances chemotherapy. We herein present the development of a novel fluorescence assay for the detection of hAGT activity. We designed a dsDNA sequence containing a fluorophore-quencher pair, where the fluorophore was attached to an O6-benzylguanine. This precursor was synthesized using the Mitsunobu reaction to introduce the benzyl group. The alkyl-fluorophore group is transferred to the active site during the dealkylation, producing an increase in fluorescence which is correlated to hAGT activity. This assay can be used for the evaluation of potential inhibitors of hAGT in a straightforward manner.The Communities MULTIFUN (contract NMP4-LA-2011-262943), Spanish Healthcare Research Fund (grant PI06/1250) and Spanish Ministry of Economy and Competitiveness (grants CTQ-2010-20541-C03-03 and CTQ2014-52588-R) are acknowledged for financialsupport. M.T. was supported by a pre-doctoral fellowship (FPI) from MINECO. C.F is grateful to TV3 Marato 2012 for a research contract.Peer reviewe

Biodegradable liposome-encapsulated hydrogels for biomedical applications: a marriage of convenience

Hydrogels are hydrophilic three-dimensional networks with demonstrated potential for medical and pharmaceutical applications. Specifically, biopolymer-based hydrogels offer certain advantages over synthetic polymers in terms of biocompatibility and biodegradability. Because of their inherent properties, hydrogels are able to efficiently encapsulate and liberate in a controlled release manner, different hydrophobic and hydrophilic therapeutic molecules, including nucleic acids, proteins and antibodies. Several strategies have been reported in the literature to minimize the potential burst release of encapsulated drugs, thus preventing their local accumulation and consequent toxic responses. Within this context, liposomes embedded in hydrogels have emerged as an attractive strategy to reduce this undesirable effect. This tutorial review covers a selection of the most promising cationic, neutral and anionic biopolymer-based hydrogels containing liposomes, niosomes or vesicles for drug delivery or tissue engineering applications.This work is supported by the University of Regensburg, the DFG (DI 1748/3-1), the Spanish Ministry of Education (Grant CTQ2014-52588-R, RTC-2014-2038-1), the Generalitat de Catalunya (2014/SGR/624), the Instituto de Salud Carlos III (CB06_01_0019) and the Spanish Ministry of Education, Culture and Sports. S.G. thanks the Spanish Ministry of Education, Culture and Sports for a “Jose Castillejo” grant within the researchers mobility program (Programa Estatal de Promoción del Talento y su Empleabilidad en I + D + i, Subprograma Estatal de Movilidad, del Plan Estatal de Investigación Científica y Técnica y de Innovación 2013–2016). D. D. D. thanks the Deutsche Forschungsgemeinschaft (DFG) for the Heisenberg Professorship Award. We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)Peer reviewe

Thioctic acid derivatives as building blocks to incorporate DNA oligonucleotides onto gold nanoparticles

Oligonucleotide gold nanoparticle conjugates are being used as diagnostic tools and gene silencing experiments. Thiol-chemistry is mostly used to functionalize gold nanoparticles with oligonucleotides and to incorporate DNA or RNA molecules onto gold surfaces. However, the stability of such nucleic acid-gold nanoparticle conjugates in certain conditions may be a limitation due to premature break of the thiol-gold bonds followed by aggregation processes. Here, we describe a straightforward synthesis of oligonucleotides carrying thioctic acid moiety based on the use of several thioctic acid-L- Threoninol derivatives containing different spacers, including triglycine, short polyethyleneglycol, or aliphatic spacers. The novel thioctic-oligonucleotides were used for the functionalization of gold nanoparticles and the surface coverage and stability of the resulting thioctic-oligonucleotide gold nanoparticles were assessed. In all cases gold nanoparticles functionalized with thioctic-oligonucleotides had higher loadings and higher stability in the presence of thiols than gold nanoparticles prepared with commercially available thiol-oligonucleotides. Furthermore, the thioctic derivative carrying the triglycine linker is sensitive to cathepsin B present in endosomes. In this way this derivative may be interesting for the cellular delivery of therapeutic oligonucleotides as these results provides the basis for a potential endosomal escape.This work is supported by the European Commission (Grants FP7-NMP-213382-2, FUNMOL and NMP4-LA-2011-262943, MULTIFUN), by the Spanish Ministry of Education (Grant CTQ2010-20541), the Generalitat de Catalunya (2009/SGR/208) and the Instituto de Salud Carlos III (CB06_01_0019). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)Peer reviewe

Stepwise synthesis of oligonucleotide-peptide conjugates containing guanidinium and lipophilic groups in their 3'-termini

Two different series of oligonucleotide-peptide conjugates have been efficiently synthesized by stepwise solid-phase synthesis. First, oligonucleotides and oligonucleotide phosphorothioates containing polar groups at the 3′-termini, such as amine and guanidinium groups were prepared. ODNs conjugates carrying several lysine residues were obtained directly from Fmoc deprotection whereas ODN conjugates with guanidinium groups were obtained by post-synthetic guanidinylation. The second family contains different urea moieties that were achieved by standard protocols. All products were fully characterized by reversed phase HPLC and MALDI-TOF mass spectrometry yielding satisfactory results. Oligonucleotide-phosphorothioate conjugates were evaluated as potential antisense oligonucleotides in the inhibition of the luciferase gene

Magnetic Gel Composites for Hyperthermia Cancer Therapy

Hyperthermia therapy is a medical treatment based on the exposition of body tissue to slightly higher temperatures than physiological (i.e., between 41 and 46 °C) to damage and kill cancer cells or to make them more susceptible to the effects of radiation and anti-cancer drugs. Among several methods suitable for heating tumor areas, magnetic hyperthermia involves the introduction of magnetic micro/nanoparticles into the tumor tissue, followed by the application of an external magnetic field at fixed frequency and amplitude. A very interesting approach for magnetic hyperthermia is the use of biocompatible thermo-responsive magnetic gels made by the incorporation of the magnetic particles into cross-linked polymer gels. Mainly because of the hysteresis loss from the magnetic particles subjected to a magnetic field, the temperature of the system goes up and, once the temperature crosses the lower critical solution temperature, thermo-responsive gels undergo large volume changes and may deliver anti-cancer drug molecules that have been previously entrapped in their networks. This tutorial review describes the main properties and formulations of magnetic gel composites conceived for magnetic hyperthermia therapy.Financial from DFG (PRJ 9209720) and University of Regensburg are gratefully acknowledged. D.D.D. thanks DFG for the Heisenberg Professorship Award.We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)Peer reviewe

Alginate Hydrogels as Scaffolds and Delivery Systems to Repair the Damaged Spinal Cord

Alginate (ALG) is a lineal hydrophilic polysaccharide present in brown algae cell walls, which turns into a gel state when hydrated. Gelation readily produces a series of three dimensional (3D) architectures like fibers, capillaries, and microspheres, used as biosensors and bio‐actuators in a plethora of biomedical applications like drug delivery and wound healing. Hydrogels have made a great impact on regenerative medicine and tissue engineering because they are able to mimic the mechanical properties of natural tissues due to their high water content. Recent advances in neurosciences have led to promising strategies for repairing and/or regenerating the damaged nervous system. Spinal cord injury (SCI) is particularly challenging, owing to its devastating medical, human, and social consequences. Although effective therapies to repair the damaged spinal cord (SC) are still lacking, multiple pharmacological, genetic, and cell‐based therapies are currently under study. In this framework, ALG hydrogels constitute a source of potential tools for the development of implants capable of promoting axonal growth and/or delivering cells or drugs at specific damaged sites, which may result in therapeutic strategies for SCI. In this mini‐review, the current state of the art of ALG applications in neural tissues for repairing the damaged spinal cord is discussed

Tuning G-quadruplex nanostructures with lipids. Towards designing hybrid scaffolds for oligonucleotide delivery

Two G-quadruplex forming oligonucleotides [d(TG4T)4 and d(TG6T)4] were selected as two tetramolecular quadruplex nanostructures because of their demonstrated ability to be modified with hydrophobic molecules. This allowed us to synthesize two series of G-quadruplex conjugates that differed in the number of G-tetrads, as well as in the terminal position of the lipid modification. Both solution and solid-phase syntheses were carried out to yield the corresponding lipid oligonucleotide conjugates modified at their 3′- and 5′-termini, respectively. Biophysical studies confirmed that the presence of saturated alkyl chains with different lengths did not affect the G-quadruplex integrity, but increased the stability. Next, the G-quadruplex domain was added to an 18-mer antisense oligonucleotide. Gene silencing studies confirmed the ability of such G-rich oligonucleotides to facilitate the inhibition of target Renilla luciferase without showing signs of toxicity in tumor cell lines