Fixation and Visualization of Full Protein Corona on Lipid Surface of Composite Nanoconstruction.

Spontaneous sorption of proteins on the nanoparticles' surface leads to the fact that nanoparticles in biological media are always enveloped by a layer of proteins-the protein corona. Corona proteins affect the properties of nanoparticles and their behavior in a biological environment. In this regard, knowledge about the composition of the corona is a necessary element for the development of nanomedicine. Because proteins have different sorption efficacy, isolating particles with a full corona and characterizing the full corona is challenging. In this study, we propose a photo-activated cross-linker for full protein corona fixation. We believe that the application of our proposed approach will make it possible to capture and visualize the full corona on nanoparticles coated with a lipid shell.

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.

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.

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.