Method of carrier-free delivery of therapeutic RNA importable into human mitochondria: Lipophilic conjugates with cleavable bonds:

Defects in mitochondrial DNA often cause neuromuscular pathologies, for which no efficient therapy has yet been developed. MtDNA targeting nucleic acids might therefore be promising therapeutic candidates. Nevertheless, mitochondrial gene therapy has never been achieved because DNA molecules can not penetrate inside mitochondria in vivo. In contrast, some small non-coding RNAs are imported into mitochondrial matrix, and we recently designed mitochondrial RNA vectors that can be used to address therapeutic oligoribonucleotides into human mitochondria. Here we describe an approach of carrier-free targeting of the mitochondrially importable RNA into living human cells. For this purpose, we developed the protocol of chemical synthesis of oligoribonucleotides conjugated with cholesterol residue through cleavable covalent bonds. Conjugates containing pH-triggered hydrazone bond were stable during the cell transfection procedure and rapidly cleaved in acidic endosomal cellular compartments. RNAs conjugated to cholesterol through a hydrazone bond were characterized by efficient carrier-free cellular uptake and partial co-localization with mitochondrial network. Moreover, the imported oligoribonucleotide designed to target a pathogenic point mutation in mitochondrial DNA was able to induce a decrease in the proportion of mutant mitochondrial genomes. This newly developed approach can be useful for a carrier-free delivery of therapeutic RNA into mitochondria of living human cells

Modeling of antigenomic therapy of mitochondrial diseases by mitochondrially addressed RNA targeting a pathogenic point mutation in mitochondrial DNA

Defects in mitochondrial genome can cause a wide range of clinical disorders, mainly neuromuscular diseases. Presently, no efficient therapeutic treatment has been developed against this class of pathologies. Because most of deleterious mitochondrial mutations are heteroplasmic, meaning that wild type and mutated forms of mitochondrial DNA (mtDNA) coexist in the same cell, the shift in proportion between mutant and wild type molecules could restore mitochondrial functions. Recently, we developed mitochondrial RNA vectors that can be used to address anti-replicative oligoribonucleotides into human mitochondria and thus impact heteroplasmy level in cells bearing a large deletion in mtDNA. Here, we show that this strategy can be also applied to point mutations in mtDNA. We demonstrate that specifically designed RNA molecules containing structural determinants for mitochondrial import and 20-nucleotide sequence corresponding to the mutated region of mtDNA, are able to anneal selectively to the mutated mitochondrial genomes. After being imported into mitochondria of living human cells in culture, these RNA induced a decrease of the proportion of mtDNA molecules bearing a pathogenic point mutation in the mtDNA ND5 gene

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

Нанотрубки со структурой типа «магнитное ядро-оболочка из благородного металла»

Using a simple two-stage method including the electrochemical synthesis of Ni nanotubes in the pores of PET membranes and their coating with gold or platinum, nanotubes with a structure of the «magnetic core - noble metal shell» type have been synthesized. The morphology of the coating is a thin continuous film with growths of various shapes. X-ray diffraction analysis estimated separate phases of nickel (core) and noble metal (coating). The magnetic properties of coated nanotubes do not differ significantly from the properties of the initial nanotubes. The method allows one to synthesize structures such as one-dimensional nanostructures of the «magnetic core - noble metal shell» type for use in the detection of chemical and biological compounds, as magnetic carriers for the delivery of drugs and genes, which can also be used as multicyclic catalysts on a magnetic carrier.С использованием простого двухстадийного метода, включающего электрохимический синтез Ni нанотрубок в порах ПЭТФ-мембран и их покрытие золотом или платиной химическим методом, синтезированы нанотрубки со структурой типа «магнитное ядро-оболочка из благородного металла». Морфология покрытия представляет собой тонкие постоянные пленки с наростами различной формы. Рентгеноструктурный анализ выявил отдельные фазы никеля (ядро) и благородного металла (покрытие). Магнитные свойства покрытых нанотрубок существенно не отличаются от свойств исходных нанотрубок. Метод позволяет синтезировать одномерные наноструктуры типа «магнитное ядро-оболочка из благородного металла» для применения при детектировании химических и биологических соединений, в качестве магнитных носителей при доставке лекарств и генов, а также могут быть использованы в качестве многоциклических катализаторов на магнитном носителе

Features of electron-microscopic visualization of soft phospholipid nanoparicles

Transmission electron microscopy (EM) is the way to control structure of lipid nanoparticles. Our work was aimed at study of EM imaging of nanoparticles based on mixtures of dioleoylphosphatidylcholine (DOPE) and phosphatidyl acid (PA), and phosphatidylcholine (PC) and cholesterol (Chol), using contrasting with uranyl acetate (UA), and phospho-tungstic acid (PTA). Suspensions of nanoparticles DOPE/PA (9:2) and PC/Chol (1:1) were adsorbed on formvar film, 1 min, and contrasted with UA, or PTA (pH 0.5), or PTA (pH 7.0), 5-10 sec, then studied in EM Jem-1400 (Jeol, Japan). Spectra of 31P-NMR of samples were recorded on spectrometer AV-300 (Bruker, Germany). Nanoparticles DOPE/PA, UA and PTA (pH 0.5) contrasting: rounded particles, formed by filaments (2-3 nm) of low electron density (ED). Filament morphology corresponds to structures of inverted hexagonal phase (IHP). PTA (pH 7.0) contrasting leads to filament structuring and appearance of membrane-like structures, morphologically corresponding to lamellar phase (LP). However, 31Р-NMR analysis revealed in sample spectrum single signal on 0,189 m.d. (IHP). Nanoparticles PC/Chol, UA and PTA (pH 0.5) contrasting: irregular particles, formed by filaments (2 nm), low ED, corresponding to IHP. The PTA revealed stacks of lipid bilayers in preparation, corresponding to LP. Presence of lipid different phases is confirmed by 31Р-NMR: analysis of preparation spectra, showed LP (signal -0,482 M. D.), IHP and isotropic phase (signals on -0,235, -0,362 M. D.). Thus, EM allows to identify lipid phase state; “traditional” contrasting agents differently identify components in same preparation, suggesting their active interaction with lipid molecules, and indicates necessity of using different contrasting for visualization of lipid nanostructures

An Influence of Modification with Phosphoryl Guanidine Combined with a 2′-O-Methyl or 2′-Fluoro Group on the Small-Interfering-RNA Effect

Small interfering RNA (siRNA) is the most important tool for the manipulation of mRNA expression and needs protection from intracellular nucleases when delivered into the cell. In this work, we examined the effects of siRNA modification with the phosphoryl guanidine (PG) group, which, as shown earlier, makes oligodeoxynucleotides resistant to snake venom phosphodiesterase. We obtained a set of siRNAs containing combined modifications PG/2′-O-methyl (2′-OMe) or PG/2′-fluoro (2′-F); biophysical and biochemical properties were characterized for each duplex. We used the UV-melting approach to estimate the thermostability of the duplexes and RNAse A degradation assays to determine their stability. The ability to induce silencing was tested in cultured cells stably expressing green fluorescent protein. The introduction of the PG group as a rule decreased the thermodynamic stability of siRNA. At the same time, the siRNAs carrying PG groups showed increased resistance to RNase A. A gene silencing experiment indicated that the PG-modified siRNA retained its activity if the modifications were introduced into the passenger strand

Chemical Modifications Influence the Number of siRNA Molecules Adsorbed on Gold Nanoparticles and the Efficiency of Downregulation of a Target Protein

Small interfering RNAs (siRNAs) are a powerful tool for specific suppression of protein synthesis in the cell, and this determines the attractiveness of siRNAs as a drug. Low resistance of siRNA to nucleases and inability to enter into target cells are the most crucial issues in developing siRNA-based therapy. To face this challenge, we designed multilayer nanoconstruct (MLNC) with AuNP core bearing chemically modified siRNAs. We applied chemical modifications 2′-OMe and 2′-F substitutions as well as their combinations with phosphoryl guanidine group in the internucleotide phosphate. The effect of modification on the efficiency of siRNA loading into nanocarriers was examined. The introduction of the internucleotide modifications into at least one of the strands raised the efficiency of siRNA adsorption on the surface of gold core. We also tested the stability of modified siRNA adsorbed on gold core in the presence of serum. Based on loading efficiency and stability, MLNCs with the most siRNA effective cargo were selected, and they showed an increase in biological activity compared to control MLNCs. Our study demonstrated the effect of chemical modifications of siRNA on its binding to the AuNP-based carrier, which directly affects the efficiency of target protein expression inhibition

A Lipid-Coated Nanoconstruct Composed of Gold Nanoparticles Noncovalently Coated with Small Interfering RNA: Preparation, Purification and Characterization

There is an urgent need to develop systems for nucleic acid delivery, especially for the creation of effective therapeutics against various diseases. We have previously shown the feasibility of efficient delivery of small interfering RNA by means of gold nanoparticle-based multilayer nanoconstructs (MLNCs) for suppressing reporter protein synthesis. The present work is aimed at improving the quality of preparations of desired MLNCs, and for this purpose, optimal conditions for their multistep fabrication were found. All steps of this process and MLNC purification were verified using dynamic light scattering, transmission electron microscopy, and UV-Vis spectroscopy. Factors influencing the efficiency of nanocomposite assembly, colloidal stability, and purification quality were identified. These data made it possible to optimize the fabrication of target MLNCs bearing small interfering RNA and to substantially improve end product quality via an increase in its homogeneity and a decrease in the amount of incomplete nanoconstructs. We believe that the proposed approaches and methods will be useful for researchers working with lipid nanoconstructs