Double-tailed lipid modification as a promising candidate for oligonucleotide delivery in mammalian cells

Background The potential use of nucleic acids as therapeutic drugs has triggered the quest for oligonucleotide conjugates with enhanced cellular permeability. To this end, the biophysical aspects of previously reported potential lipid oligodeoxyribonucleotide conjugates were studied including its membrane-binding properties and cellular uptake. Methods These conjugates were fully characterized by MALDI-TOF mass spectrometry and HPLC chromatography. Their ability to insert into lipid model membrane systems was evaluated by Langmuir balance and confocal microscopy followed by the study of the internalization of a lipid oligodeoxyribonucleotide conjugate bearing a double-tail lipid modification (C28) into different cell lines by confocal microscopy and flow cytometry. This compound was also compared with other lipid containing conjugates and with the classical lipoplex formulation using Transfectin as transfection reagent. Results This double-tail lipid modification showed better incorporation into both lipid model membranes and cell systems. Indeed, this lipid conjugation was capable of inserting the oligodeoxyribonucleotide into both liquid-disordered and liquid-ordered domains of model lipid bilayer systems and produced an enhancement of oligodeoxyribonucleotide uptake in cells, even better than the effect caused by lipoplexes. In addition, in β2 integrin (CR3) expressing cells this receptor was directly involved in the enhanced internalization of this compound. Conclusions All these features confirm that the dual lipid modification (C28) is an excellent modification for enhancing nucleic acid delivery without altering their binding properties. General significance Compared to the commercial lipoplex approach, oligodeoxyribonucleotide conjugation with C28 dual lipid modification seems to be promising to improve oligonucleotide delivery in mammalian cells.This work was supported with funds from the Spanish Ministry of Economy [Grant BFU2007-62062], the Basque Government [GIV06-42], the Spanish Ministry of Education [Grant CTQ2010-20541], the Generalitat de Catalunya [2009/SGR/208], the Instituto de Salud Carlos III [CB06_01_0019] and Fundación Biofísica Bizkaia. CIBER-BBN is an iniciative 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. B.U-U. was supported by Universidad de País Vasco-UPV/EHU pre-doctoral fellowship and Fundación Biofísica Bizkaia. J.V.B. was a postdoctoral scientist supported by Universidad de País Vasco-UPV/EHU postdoctoral fellowship. The authors acknowledge the Servicio General de Microscopía Analítica y de Alta Resolución en Biomedicina at the University of Basque Country for assistance with confocal microscopy, Prof. A. Gómez-Muñoz for flow cytometry facilities and Eneritz Bilbao for excellent technical assistance.Peer reviewe

Delivery of small nucleic acids by conjugation to carbohydrates and lipids as novel research and therapeutic tools

399 p. : il., graf.Small nucleic acids present a great potential to silence specific genes or inhibit the biological activity of specific proteins. Unfortunately, their poor cellular accessibility presents a big challenge for developing an efficient delivery system. In this regard, conjugation of appropriate molecules to small nucleic improves their pharmacokinetic behaviours and cellular uptake efficiencies, endowing them with entirely new properties. In the present work different carbohydrate- and lipid-oligonucleotide conjugates have been studied as novel tools for targeted delivery and enhancement of cellular permeability. The results obtained in this work indicate that keeping a certain distance between DNA and sugar modification could be important for a better incorporation of this type of conjugates into the target cell. Also long or double-tailed lipid modifications are preferred for an enhanced incorporation of lipid-oligonucleotide conjugates into membrane-model and cell systems.The present thesis has been performed Departamento de Bioquímica y Biología Molecular de la Universidad del País Vasco and Unidad de Biofísica (Centro Mixto CSIC-UPV/EHU). This work has been supported mainly by CSIC (CARBINH, PIF06-045). The author received a research fellowship from the University of the Basque Country (PIFA01/2006/052, June 2007-May 2011) and a research contract supported by Fundación Biofísica Bizkaia (June 2011-May 2012)

Carrier-free cellular uptake and the gene-silencing activity of the lipophilic siRNAs is strongly affected by the length of the linker between siRNA and lipophilic group

The conjugation of siRNA to molecules, which can be internalized into the cell via natural transport mechanisms, can result in the enhancement of siRNA cellular uptake. Herein, the carrier-free cellular uptake of nuclease-resistant anti-MDR1 siRNA equipped with lipophilic residues (cholesterol, lithocholic acid, oleyl alcohol and litocholic acid oleylamide) attached to the 5′-end of the sense strand via oligomethylene linker of various length was investigated. A convenient combination of H-phosphonate and phosphoramidite methods was developed for the synthesis of 5′-lipophilic conjugates of siRNAs. It was found that lipophilic siRNA are able to effectively penetrate into HEK293, HepG2 and KB-8-5 cancer cells when used in a micromolar concentration range. The efficiency of the uptake is dependent upon the type of lipophilic moiety, the length of the linker between the moiety and the siRNA and cell type. Among all the conjugates tested, the cholesterol-conjugated siRNAs with linkers containing from 6 to 10 carbon atoms demonstrate the optimal uptake and gene silencing properties: the shortening of the linker reduces the efficiency of the cellular uptake of siRNA conjugates, whereas the lengthening of the linker facilitates the uptake but retards the gene silencing effect and decreases the efficiency of the silencing

СИНТЕЗ КОНЪЮГАТОВ ОЛИГОНУКЛЕОТИДОВ С ПРОИЗВОДНЫМИ 11,11-D2-ЛИНОЛЕВОЙ КИСЛОТЫ ПО РЕАКЦИИ АЗИД-АЛКИНОВОГО ЦИКЛОПРИСОЕДИНЕНИЯ

A new approach to the synthesis of oligonucleotide-fatty acid conjugates is reported. It is based on Cu-catalyzed reaction between azide oligonucleotides and fatty acid derivatives with terminal triple bond (CuAAC). To demonstrate this approach, five derivatives of 11,11-D2-linoleic acid containing terminal alkyne group at different parts of the molecule were synthesized. A method of conjugation of alkyne 11,11-D2-linoleic acid with azide oligonucleotide T20 is developed, and the conditions (time, concentration of Cu catalyst, excess of alkyne reagent, composition of the solvent, etc.) are optimized. These conjugates are stable in biological media, have increased permeability through the cell membranes and can be used in gene therapy. Предложен новый подход к синтезу конъюгатов олигонуклеотидов и жирных кислот, основанный на медь-катализируемой реакции циклоприсоединения азидсодержащих олигонуклеотидов к производным жирных кислот с терминальной тройной связью (CuAAC). Для демонстрации этого подхода осуществлен синтез пяти производных 11,11-D2-линолевой кислоты, содержащих концевую тройную связь в различных частях молекулы. Разработана методика их конъюгации с азидсодержащим модельным олигонуклеотидом Т20, проверено влияние различных условий (времени, концентрации катализатора, избытка алкинсодержащего реагента, состава растворителя и др.) на выход продукта. Такие конъюгаты устойчивы в биологических средах, обладают повышенной способностью проникать в клетки и могут найти применение в качестве средств генной терапии.

Covalent conjugates of therapeutic oligonucleotides for in vivo targeting

Over last few years, the development of oligonucleotide based therapeutics (antisense, siRNA, antagomirs) have received much interest as a novel class of drugs for the treatment of many diseases. Cell/organ specific targeting of oligonucleotides by covalent conjugation has become a promising approach for developing therapeutic RNAs. The major obstacle in the use of therapeutic RNAs is the cell/organ specific targeting and internalization of the large anionic oligonucleotides across the plasma membrane of the cells. This thesis focuses on the synthesis of different receptor specific ligand conjugates of oligonucleotides. The oligonucleotides are conjugated with different targeting ligands such as i) Galactose cluster, ii) Hyaluronic acid hexamer, and iii) Bisphosphonate derivative. Multi-galactose-conjugated 2’-O-methyl oligoribonucleotides showed remarkable galactose-dependent liver targeting of the conjugates monitored by in vivo positron emission tomography (PET) imaging in healthy rats. Hyaluronic acid hexasaccharide oligonucleotide were conjugated efficiently by using copper free click reaction (SPAAC conjugation approach) and also studied by in vivo PET imaging in myocardial infarction rat models. Bone targeting bisphosphonate oligonucleotide conjugates were prepared by SPAAC conjugation approach and in vivo PET imaging exhibited enriched radioactivity accumulation to bones in healthy rats. Additionally, a straightforward method was described for the synthesis of solid supported porphyrin biomolecule conjugates. The whole-body distribution of the conjugates in rats was monitored by PET. These oligonucleotide conjugates were efficiently labeled by complexing 68Ga, with a 3´-terminal 1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid (NOTA) ligand. This allowed in vivo quantification of oligonucleotide pharmacokinetics and bio-distribution data in rats.Siirretty Doriast

Nanotechnology and the Treatment of HIV Infection

Suboptimal adherence, toxicity, drug resistance and viral reservoirs make the lifelong treatment of HIV infection challenging. The emerging field of nanotechnology may play an important role in addressing these challenges by creating drugs that possess pharmacological advantages arising out of unique phenomena that occur at the “nano” scale. At these dimensions, particles have physicochemical properties that are distinct from those of bulk materials or single molecules or atoms. In this review, basic concepts and terms in nanotechnology are defined, and examples are provided of how nanopharmaceuticals such as nanocrystals, nanocapsules, nanoparticles, solid lipid nanoparticles, nanocarriers, micelles, liposomes and dendrimers have been investigated as potential anti-HIV therapies. Such drugs may, for example, be used to optimize the pharmacological characteristics of known antiretrovirals, deliver anti-HIV nucleic acids into infected cells or achieve targeted delivery of antivirals to the immune system, brain or latent reservoirs. Also, nanopharmaceuticals themselves may possess anti-HIV activity. However several hurdles remain, including toxicity, unwanted biological interactions and the difficulty and cost of large-scale synthesis of nanopharmaceuticals

From Poly(Alkyl Cyanoacrylate) to Squalene as Core Material to Design Nanomedicines

International audienceThe discovery of biodegradable poly(alkyl cyanoacrylate) (PACA) nanoparticles by Patrick Couvreur has opened large perspectives in nanomedicine. Nanoparticles made from different types of PACA monomers have been used in different applications such as the treatment of intracellular infections or the treatment of multidrug resistant hepatocarcinoma. This latest application led to a Phase 3 clinical trial of Livatag ® , a PACA nanoparticulate formulation of doxorubicin. Despite the success of PACA nanoparticles, the need to develop novel type of nanoparticles with higher drug loadings and lower burst release was tackled by the discovery of squalene-based nanoparticles where the drug is covalently linked to the lipid derivative and the resulting conjugate self-assemble into nanoparticles. This pioneering work was accompanied by a wide range of novel applications which mainly dealt with the management of unmet medical needs (e.g., pancreatic cancer, brain ischemia and spinal cord injury). The present Review Article covers the most important steps of the pioneering work of Patrick Couvreur by trying to shed light on his outstanding career that has been a source of inspiration for many decades

Dendrimers and derivatives as a potential therapeutic tool in regenerative medicine strategies : a review

Since the pioneering work dealing with the synthesis and physicochemical aspects of dendrimers, a predictable and tunable set of compositions for therapeutic, scaffolding and imaging systems has been reported. These are well documented, butmanyhot issues should be examined and reviewed. Herein, a review is given on dendritic nanopolymers and their applications that show promise in the field of regenerative medicine. This review begins with a brief overview on research merging nanotechnology and regenerative medicine. Fundamentals of the synthesis and macromolecular structure of dendritic polymers are provided. Dendrimers fulfill the requirements as carriers for gene, nucleic acids, bioactive molecules and peptide/protein delivery aimed at modulate the cells functions, in vitro and in vivo. However, to make use of this potential, toxicological, drug-loading capacity, surface engineering and host–guest chemistries in dendrimers must be addressed and thus are also discussed. We focus on recent work involving dendrimers with applications in tissue engineering and the central nervous system. Due to their innovative character, applications beyond drug delivery systems became possible, namely as scaffolding and chemoattractants for tissue regeneration, and implantable biodegradable nanomaterialbased medical devices integrated with drug delivery functions (theranostics). Finally, we highlight promising areas for further research and comment on how and why dendrimer and dendron technology should be viewed as the next generation of biomaterials for the 21st century.The authors wish to acknowledge the funding provided by the Portuguese Foundation for Science and Technology (FCT) through the POCTI and FEDER programs, and FCT project (SMARTCARBO). This work was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283) and European Union HIPPOCRATES STREP Project (NMP3-CT-2003-505758). Canon Foundation in Europe is also gratefully acknowledged

Synthetic materials at the forefront of gene delivery

This is the Author's Accepted Manuscript of the following article: Lostalé-Seijo, I., & Montenegro, J. (2018). Synthetic materials at the forefront of gene delivery. Nature Reviews Chemistry. doi: 10.1038/s41570-018-0039-1The delivery of nucleic acids with transient activity for genetic engineering is a promising methodology with potential applications in the treatment of diseases ranging from cancer and infectious diseases to heritable disorders. Restoring the expression of a missing protein, correcting defective splicing of transcripts and silencing or modulating the expression of genes are powerful approaches that could have substantial benefits in biological research and medicine. Impressive progress in improving gene delivery has been made in the past decade, and several products have reached the market. However, translating the results of in vitro and preclinical studies into functional therapies is hindered by the suboptimal performance of gene delivery vehicles in capturing, protecting and delivering nucleic acid cargoes safely and efficaciously. Chemistry has a key role in the development of innovative synthetic materials to overcome the challenges of producing next-generation gene delivery therapies and protocols. In this Review, we discuss the latest chemical advances in the production of materials for the delivery of nucleic acids to cells and for gene therapyThe group of J.M. was partially supported by the Spanish Agencia Estatal de Investigación (AEI) (CTQ2014-59646-R and SAF2017-89890-R), the Xunta de Galicia (ED431G/09, ED431C 2017/25 and 2016-AD031) and the European Regional Development Fund (ERDF). J.M. received a Ramón y Cajal grant (RYC-2013-13784), a European Research Council (ERC) Starting Investigator Grant (DYNAP-677786) and a Young Investigator Grant from the Human Frontier Science Research Program (RGY0066/2017)S