Complexes and Supramolecular Associates of Dodecyl-Containing Oligonucleotides with Serum Albumin.

Serum albumin is currently in the focus of biomedical research as a promising platform for the creation of multicomponent self-assembling systems due to the presence of several sites with high binding affinity of various compounds in its molecule, including lipophilic oligonucleotide conjugates. In this work, we investigated the stoichiometry of the dodecyl-containing oligonucleotides binding to bovine and human serum albumins using an electrophoretic mobility shift assay. The results indicate the formation of the albumin-oligonucleotide complexes with a stoichiometry of about 1 : (1.25 ± 0.25) under physiological-like conditions. Using atomic force microscopy, it was found that the interaction of human serum albumin with the duplex of complementary dodecyl-containing oligonucleotides resulted in the formation of circular associates with a diameter of 165.5 ± 94.3 nm and 28.9 ± 16.9 nm in height, and interaction with polydeoxyadenylic acid and dodecyl-containing oligothymidylate resulted in formation of supramolecular associates with the size of about 315.4 ± 70.9 and 188.3 ± 43.7 nm, respectively. The obtained data allow considering the dodecyl-containing oligonucleotides and albumin as potential components of the designed self-assembling systems for solving problems of molecular biology, biomedicine, and development of unique theranostics with targeted action.

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.

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.

Effect of Fluorescent Labels on DNA Affinity for Gold Nanoparticles.

Fluorophore (FD) labeling is widely used for detection and quantification of various compounds bound to nanocarriers. The systems, composed of gold nanoparticles (GNPs) and oligonucleotides (ONs) labeled with FDs, have wide applications. Our work was aimed at a systemic study of how FD structure (in composition of ON-FDs) influenced the efficiency of their non-covalent associates' formation with GNPs (ON-FD/GNPs). We examined ONs of different length and nucleotide composition, and corresponding ON-FDs (FDs from a series of xanthene, polymethine dyes; dyes based on polycyclic aromatic hydrocarbons). Methods: fluorometry, dynamic light scattering, high performance liquid chromatography, gel electrophoresis, molecular modeling and methods of thermodynamic and statistical analysis. We observed significant, differing several times, changes in surface density and Langmuir constant values of ON-FDs vs. ONs, evidence for the critical significance of FD nature for binding of ON-FDs with GNPs. Surface density of ON-FD/GNPs; hydrophobicity and total charge of ON or ON-FD; and charge and surface area of FDs were revealed as key factors determining affinity (Langmuir constant) of ON or ON-FDs for GNPs. These factors compose a specific set, which makes possible the highly reliable prediction of efficiency of ONs and ON-FDs binding with GNPs. The principal possibility of creating an algorithm for predictive calculation of efficiency of ONs and GNPs interaction was demonstrated. We proposed a hypothetical model that described the mechanism of contact interaction between negatively charged nano-objects, such as citrate-stabilized GNPs, and ONs or ON-FDs.

Isolation of Extracellular Vesicles from Biological Fluids via the Aggregation-Precipitation Approach for Downstream miRNAs Detection.

Extracellular vesicles (EVs) have high potential as sources of biomarkers for non-invasive diagnostics. Thus, a simple and productive method of EV isolation is demanded for certain scientific and medical applications of EVs. Here we aim to develop a simple and effective method of EV isolation from different biofluids, suitable for both scientific, and clinical analyses of miRNAs transported by EVs. The proposed aggregation-precipitation method is based on the aggregation of EVs using dextran blue and the subsequent precipitation of EVs using 1.5% polyethylene glycol solutions. The developed method allows the effective isolation of EVs from plasma and urine. As shown using TEM, dynamic light scattering, and miRNA analyses, this method is not inferior to ultracentrifugation-based EV isolation in terms of its efficacy, lack of inhibitors for polymerase reactions and applicable for both healthy donors and cancer patients. This method is fast, simple, does not need complicated equipment, can be adapted for different biofluids, and has a low cost. The aggregation-precipitation method of EV isolation accessible and suitable for both research and clinical laboratories. This method has the potential to increase the diagnostic and prognostic utilization of EVs and miRNA-based diagnostics of urogenital pathologies.

Delivery of mRNA Vaccine against SARS-CoV-2 Using a Polyglucin:Spermidine Conjugate.

One of the key stages in the development of mRNA vaccines is their delivery. Along with liposome, other materials are being developed for mRNA delivery that can ensure both the safety and effectiveness of the vaccine, and also facilitate its storage and transportation. In this study, we investigated the polyglucin:spermidine conjugate as a carrier of an mRNA-RBD vaccine encoding the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The conditions for the self-assembling of mRNA-PGS complexes were optimized, including the selection of the mRNA:PGS charge ratios. Using dynamic and electrophoretic light scattering it was shown that the most monodisperse suspension of nanoparticles was formed at the mRNA:PGS charge ratio equal to 1:5. The average hydrodynamic particles diameter was determined, and it was confirmed by electron microscopy. The evaluation of the zeta potential of the investigated complexes showed that the particles surface charge was close to the zero point. This may indicate that the positively charged PGS conjugate has completely packed the negatively charged mRNA molecules. It has been shown that the packaging of mRNA-RBD into the PGS envelope leads to increased production of specific antibodies with virus-neutralizing activity in immunized BALB/c mice. Our results showed that the proposed polycationic polyglucin:spermidine conjugate can be considered a promising and safe means to the delivery of mRNA vaccines, in particular mRNA vaccines against SARS-CoV-2.

Rational Design of Albumin Theranostic Conjugates for Gold Nanoparticles Anticancer Drugs: Where the Seed Meets the Soil?

Multifunctional gold nanoparticles (AuNPs) may serve as a scaffold to integrate diagnostic and therapeutic functions into one theranostic system, thereby simultaneously facilitating diagnosis and therapy and monitoring therapeutic responses. Herein, albumin-AuNP theranostic agents have been obtained by conjugation of an anticancer nucleotide trifluorothymidine (TFT) or a boron-neutron capture therapy drug undecahydro-closo-dodecaborate (B12H12) to bimodal human serum albumin (HSA) followed by reacting of the albumin conjugates with AuNPs. In vitro studies have revealed a stronger cytotoxicity by the AuNPs decorated with the TFT-tagged bimodal HSA than by the boronated albumin conjugates. Despite long circulation time, lack of the significant accumulation in the tumor was observed for the AuNP theranostic conjugates. Our unique labelling strategy allows for monitoring of spatial distribution of the AuNPs theranostic in vivo in real time with high sensitivity, thus reducing the number of animals required for testing and optimizing new nanosystems as chemotherapeutic agents and boron-neutron capture therapy drug candidates.

Amphiphilic "Like-a-Brush" Oligonucleotide Conjugates with Three Dodecyl Chains: Self-Assembly Features of Novel Scaffold Compounds for Nucleic Acids Delivery.

The conjugation of lipophilic groups to oligonucleotides is a promising approach for improving nucleic acid-based therapeutics' intracellular delivery. Lipid oligonucleotide conjugates can self-aggregate in aqueous solution, which gains much attention due to the formation of micellar particles suitable for cell endocytosis. Here, we describe self-association features of novel "like-a-brush" oligonucleotide conjugates bearing three dodecyl chains. The self-assembly of the conjugates into 30-170 nm micellar particles with a high tendency to aggregate was shown using dynamic light scattering (DLS), atomic force (AFM), and transmission electron (TEM) microscopies. Fluorescently labeled conjugates demonstrated significant quenching of fluorescence intensity (up to 90%) under micelle formation conditions. The conjugates possess increased binding affinity to serum albumin as compared with free oligonucleotides. The dodecyl oligonucleotide conjugate and its duplex efficiently internalized and accumulated into HepG2 cells' cytoplasm without any transfection agent. It was shown that the addition of serum albumin or fetal bovine serum to the medium decreased oligonucleotide uptake efficacy (by 22.5-36%) but did not completely inhibit cell penetration. The obtained results allow considering dodecyl-containing oligonucleotides as scaffold compounds for engineering nucleic acid delivery vehicles.

Structural and Aggregation Features of a Human κ-Casein Fragment with Antitumor and Cell-Penetrating Properties.

Intrinsically disordered proteins play a central role in dynamic regulatory and assembly processes in the cell. Recently, a human κ-casein proteolytic fragment called lactaptin (8.6 kDa) was found to induce apoptosis of human breast adenocarcinoma MCF-7 and MDA-MB-231 cells with no cytotoxic activity toward normal cells. Earlier, we had designed some recombinant analogs of lactaptin and compared their biological activity. Among these analogs, RL2 has the highest antitumor activity, but the amino acid residues and secondary structures that are responsible for RL2's activity remain unclear. To elucidate the structure-activity relations of RL2, we studied the structural and aggregation features of this fairly large intrinsically disordered fragment of human milk κ-casein by a combination of physicochemical methods: NMR, paramagnetic relaxation enhancement (PRE), Electron Paramagnetic Resonance (EPR), circular dichroism, dynamic light scattering, atomic force microscopy, and a cytotoxic activity assay. It was found that in solution, RL2 exists as stand-alone monomeric particles and large aggregates. Whereas the disulfide-bonded homodimer turned out to be more prone to assembly into large aggregates, the monomer predominantly forms single particles. NMR relaxation analysis of spin-labeled RL2 showed that the RL2 N-terminal region, which is essential not only for multimerization of the peptide but also for its proapoptotic action on cancer cells, is more ordered than its C-terminal counterpart and contains a site with a propensity for α-helical secondary structure.

Long-term stability and scale-up of noncovalently bound gold nanoparticle-siRNA suspensions.

Gold nanoparticles (AuNPs) are a platform for the creation of nanoconstructions that can have a variety of functions, including the delivery of therapeutic nucleic acids. We previously designed a AuNP/small interfering RNA (siRNA) nanoconstruction consisting of siRNA noncovalently bound on the AuNP surface and showed that this construction, when coated with a lipid shell, was an efficient vehicle for the delivery of siRNA into cells. The goal of the present work was to study the possibility of scaling up the synthesis of AuNP-siRNA and its long-term storage without loss of physicochemical characteristics and siRNA duplex integrity as well as siRNA surface density. Dynamic light scattering, transmission electron microscopy, UV-vis spectroscopy, and electrophoresis were used to study the effect of scaling up the AuNP-siRNA synthesis and long term storage of its suspension on physicochemical properties of the samples and integrity of the siRNA duplex. It was shown that a ten-fold increase in the volume of the reaction mixture decreased the surface density of siRNA by about 10%, which influenced the corresponding physicochemical characteristics of the AuNP-siRNA suspension. The storage of the AuNP-siRNA suspension at 4 °C for different times resulted in the formation of particle clusters of high colloidal stability as demonstrated by conventional methods. These clusters completely disintegrated when albumin was added, indicating that they are agglomerates (and not aggregates) of AuNP-siRNA. The AuNPs-siRNA nanoconstruction demonstrated integrity of the siRNA duplex and high stability of the siRNA surface density during storage for seven months at 4 °C. Thus, it can be concluded that it is possible to scale-up the synthesis of noncovalent AuNP-siRNA and to obtain a nanoconstruction possessing high stability in terms of physicochemical characteristics and siRNA surface density for a long period.

Colloidal FeIII , MnIII , CoIII , and CuII Hydroxides Stabilized by Starch as Catalysts of Water Oxidation Reaction with One Electron Oxidant Ru(bpy)33.

Colloidal catalysts for oxidation of water to dioxygen, which are stable on storage and under the reaction conditions, are synthesized based on CoIII , MnIII , FeIII and CuII hydroxides. Stabilization of the colloids with dextrated starch allows the process of hydroxide ageing to be stopped at the stage of the formation of primary nuclei (ca. 2-3 nm from transmission electron microscopy data). Molecular mechanics and dynamic light scattering studies indicate a core-shell type structure of the catalysts, where the hydroxide core is stabilized by the molecular starch network (ca. 5-7 nm). The colloidal catalysts are highly efficient in oxidizing water with one electron oxidant Ru(bpy)33+ at pH 7 to 10. The influence of pH, catalyst concentration, and buffer nature on the oxygen yield is studied. The maximal yields are 72, 53, and 78 % over Fe-, Mn- and Co-containing catalysts, respectively, and turnover numbers are 7.8; 54 and 360, respectively. The Cu-containing catalyst is poorly effective to the water oxidation (the maximal yield is 28 % O2 ). The synthesized catalysts are of interest for stopped-flow kinetic studies of the mechanism of the water oxidation and as precursors for anchoring nanosized hydroxides onto various supports in order to develop biomimetic systems for artificial photosynthesis.

Non-agglomerated silicon-organic nanoparticles and their nanocomplexes with oligonucleotides: synthesis and properties.

The development of efficient and convenient systems for the delivery of nucleic-acid-based drugs into cells is an urgent task. А promising approach is the use of various nanoparticles. Silica nanoparticles can be used as vehicles to deliver nucleic acid fragments into cells. In this work, we developed a method for the synthesis of silicon-organic (Si-NH2) non-agglomerated nanoparticles by the hydrolysis of aminopropyltriethoxysilane (APTES). The resulting product forms a clear solution containing nanoparticles in the form of low molecular weight polymer chains with [─Si(OH)(C3H6NH2)O─] monomer units. Oligonucleotides (ODN) were conjugated to the prepared Si-NH2 nanoparticles using the electrostatic interaction between positively charged amino groups of nanoparticles and negatively charged internucleotide phosphate groups in oligonucleotides. The Si-NH2 nanoparticles and Si-NH2·ODN nanocomplexes were characterized by transmission electron microscopy, atomic force microscopy and IR and electron spectroscopy. The size and zeta potential values of the prepared nanoparticles and nanocomplexes were evaluated. Oligonucleotides in Si-NH2·ODN complexes retain their ability to form complementary duplexes. The Si-NH2 Flu nanoparticles and Si-NH2·ODNFlu nanocomplexes were shown by fluorescence microscopy to penetrate into human cells. The Si-NH2 Flu nanoparticles predominantly accumulated in the cytoplasm whereas ODNFlu complexes were predominantly detected in the cellular nuclei. The Si-NH2·ODN nanocomplexes demonstrated a high antisense activity against the influenza A virus in a cell culture at a concentration that was lower than their 50% toxic concentration by three orders of magnitude.

Size-Dependent Ability of Liposomes to Accumulate in the Ischemic Myocardium and Protect the Heart.

Liposomes have the potential to be used for drug delivery. Meanwhile, liposome size may affect their accumulation in the target tissue. We investigated the myocardial accumulation of 2 populations of liposomes (∼70 and 110 nm diameter) during ischemia and their effect on ischemia/reperfusion injury. Isolated rat hearts were subjected to 30 minutes of low-flow ischemia with the liposomes, followed by 30 minutes of liposome-free reperfusion. The liposomes were loaded with the fluorescent dye Nile Red to assess their accumulation in the myocardium. The cardiac functional recovery during reperfusion was evaluated using force-velocity characteristics and coronary flow (CF). Reperfusion injury was evaluated by lactate dehydrogenase release. In addition, CF and contractility were assessed in hearts perfused normally with 70 nm liposomes. There was a 6- and 4-fold greater accumulation of the small liposomes in the myocardium and mitochondria, respectively, compared with the large liposomes. Importantly, even without any incorporated drugs, both populations of liposomes improved functional recovery and reduced lactate dehydrogenase release. However, the smaller liposomes showed significantly higher protective and vasodilatory effects during reperfusion than the larger particles. These liposomes also increased CF and contractility during normal perfusion. We suggest that the protective properties of the liposomes could be related to their membrane-stabilizing effect.

SDS-PAGE procedure: Application for characterization of new entirely uncharged nucleic acids analogs.

SDS-PAGE is considered to be a universal method for size-based separation and analysis of proteins. In this study, we applied the principle of SDS-PAGE to the analysis of new entirely uncharged nucleic acid (NA) analogues, - phosphoryl guanidine oligonucleotides (PGOs). The procedure was also shown to be suitable for morpholino oligonucleotides (PMOs) and peptide nucleic acids (PNAs). It was demonstrated that SDS can establish hydrophobic interactions with these types of synthetic NAs, giving them a net negative charge and thus making these molecules mobile in polyacrylamide slab gels under the influence of an electric field.

Surprises of electron microscopic imaging of proteins and polymers covering gold nanoparticles layer by layer.

Gold nanoparticles (GNPs) are used in complicated nanoconstructions, and their preparation implies careful analysis of the intermediate and resulting products, including visualisation of the NPs. Visualisation of protein and/or organic polymer covers on GNPs using electron microscopy (EM) was a goal of this study. We covered GNPs with human serum albumin or PEG, and then added a second layer of branched or linear polyethyleneimine. EM studies were supplemented with dynamic light scattering, spectrophotometry and gel electrophoresis, which confirmed the presence and integrity of a cover on GNPs in mixtures with uranylacetate (UA) or phosphotungstic acid (PTA). Covered GNPs were contrasted 'on a drop' or in suspension with UA (pH 4.5) or PTA (pH 0.5, 3.0, 5.0 and 7.0), and studied by transmission EM. A cover on GNPs becomes visible as the result of direct interaction of UA or PTA with the components of a layer. The same NPs could look 'naked' or demonstrate a distinct cover of average electron density. The most distinct images of the layers were obtained using PTA at pH 0.5. Thus, visualisation of protein and/or polymeric layers covering the GNPs by EM depends on the type of contrasting reagent and contrasting conditions, but does not depend on surface charge of the NPs and the chemical nature of a cover.

Comparison of behaviour in different liquids and in cells of gold nanorods and spherical nanoparticles modified by linear polyethyleneimine and bovine serum albumin.

Gold nanorods (GNRs) are considered one of the most promising forms of nanoparticles for nanobiotechnology; however, the problem of their toxicity is currently not resolved. We synthesised GNRs, modified with linear polyethyleneimine (PEI-GNRs), and examined their physicochemical and some biological properties in comparison with GNRs modified with BSA and spherical gold nanoparticles (sGNPs) modified with the same agents. The influence of the buffer, cell culture media, and serum on hydrodynamic diameter and zeta potential of all GNPs was studied. Simultaneously, the size, shape, and formation of a corona were examined by transmission electron microscopy (TEM). PEI-GNRs and GNPs were nontoxic for BHK-21 and HeLa cells (MTT test). Penetration of all GNPs into BHK-21, melanoma B16, and HeLa cells was examined after 30 min, 3 h, and 24 h of incubation using TEM ultrathin sections. PEI-GNRs and PEI-sGNPs demonstrated fast and active penetration into cells by caveolin-dependent and lipid raft-mediated endocytosis and accumulated in endosomes and lysosomes. BSA-modified GNPs showed prolonged flotation and a significant delay in cell penetration. The results show that the charge of initial NPs determines penetration into cells. Thus, the designed PEI-GNRs were nontoxic and stable in cell culture media and could efficiently penetrate cells.

The influence of the non-nucleotide insert on the hybridization properties of oligonucleotides.

The effect of different non-nucleotide inserts incorporated into oligonucleotide chains on their hybridization properties was studied by the method of thermal denaturation. Various types of alkyldiols and oligoethylene glycols were used as inserts modifying oligonucleotide backbone. Such modification of oligonucleotides caused the destabilization of their complementary complexes. It was shown that the hybridization properties of the modified oligonucleotides depend on several features of inserts: the type, number, length of insertions, and positions of interrupted dinucleotide steps in oligonucleotide chain.

Site-specific cleavage of RNA and DNA by complementary DNA--bleomycin A5 conjugates.

Bleomycin displays clinical chemotherapeutic activity, but is so nonspecifically toxic that it is rarely administered. It was therefore of interest to determine whether bleomycin could be directed to cleave RNA or DNA at a specific site by conjugation to a complementary oligonucleotide. A 15 nt MYC complementary oligodeoxynucleotide (HMYC55) bearing a 5' bleomycin A5 (Blm) residue was designed to base-pair with nt 7047-7061 of human MYC mRNA. Reactivity of the Blm-HMYC55 conjugate (and mismatch controls) with a MYC mRNA 30-mer, a MYC DNA 30-mer, and a MYC 2'-O-methyl RNA 30-mer, nt 7041-7070, was analyzed in 100 microM FeNH(4)SO(4), 50 mM beta-mercaptoethanol, 200 mM LiCl, 10 mM Tris-HCl, pH 7.5, at 37 degrees C. Cleavage of the substrate RNA or DNA occurred primarily at the junction of the complementary DNA-target RNA duplex, 18-22 nt from the 5' end of the RNA. Reaction products with lower mobility than the target RNA or DNA also formed. Little or no reaction was observed with more than three mismatches in a Blm-oligodeoxynucleotide conjugate. Neither the short RNA or DNA cleavage fragments nor the low mobility products were observed in the absence of Fe(II), or the presence of excess EDTA. The target RNA was also cleaved efficiently by bleomycin within a hybrid duplex with a preformed single-nucleotide bulge in the RNA strand. New Blm-oligodeoxynucleotide conjugates containing long hexaethylene glycol phosphate based linkers between oligodeoxynucleotide and bleomycin were designed to target this bulge region. These conjugates achieved 8-18% cleavage of the target RNA, depending on the length of the linker. Blm-oligodeoxynucleotide conjugates thus demonstrated sequence specificity and site specificity against RNA and DNA targets.