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

A Lipid-Coated Nanoconstruct Composed of Gold Nanoparticles Noncovalently Coated with Small... nanomaterials Article A Lipid-Coated Nanoconstruct Composed of Gold Nanoparticles Noncovalently Coated with Small Interfering RNA: Preparation, Purification and Characterization † † † Anna V. Epanchintseva , Julia E. Poletaeva , Ilya S. Dovydenko , Boris P. Chelobanov, Dmitrii V. Pyshnyi , Elena I. Ryabchikova * and Inna A. Pyshnaya * Institute of Chemical Biology and Fundamental Medicine SB RAS, 630090 Novosibirsk, Russia; annaepanch@niboch.nsc.ru (A.V.E.); fabaceae@yandex.ru (J.E.P.); dovydenko.il@gmail.com (I.S.D.); boris.p.chelobanov@gmail.com (B.P.C.); pyshnyi@niboch.nsc.ru (D.V.P.) * Correspondence: lenryab@yandex.ru (E.I.R.); pyshnaya@niboch.nsc.ru (I.A.P.); Tel.: +7-(383)-363-5163 (E.I.R. & I.A.P.) † These authors contributed equally to this work. Abstract: 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 Citation: Epanchintseva, A.V.; for their multistep fabrication were found. All steps of this process and MLNC purification were Poletaeva, J.E.; Dovydenko, I.S.; verified using dynamic light scattering, transmission electron microscopy, and UV-Vis spectroscopy. Chelobanov, B.P.; Pyshnyi, D.V.; Ryabchikova, E.I.; Pyshnaya, I.A. A Factors influencing the efficiency of nanocomposite assembly, colloidal stability, and purification Lipid-Coated Nanoconstruct quality were identified. These data made it possible to optimize the fabrication of target MLNCs Composed of Gold Nanoparticles bearing small interfering RNA and to substantially improve end product quality via an increase in Noncovalently Coated with Small its homogeneity and a decrease in the amount of incomplete nanoconstructs. We believe that the Interfering RNA: Preparation, proposed approaches and methods will be useful for researchers working with lipid nanoconstructs. Purification and Characterization. Nanomaterials 2021, 11, 2775. https:// Keywords: gold nanoparticles; siRNA; noncovalent adsorption; lipid enveloping; multilayer nanocon- doi.org/10.3390/nano11112775 structs for siRNA preparation and purification Academic Editor: Paulo Cesar De Morais 1. Introduction Received: 1 October 2021 Accepted: 18 October 2021 Nucleic-acid therapeutics have tremendous therapeutic potential for the treatment of Published: 20 October 2021 many diseases. Nonetheless, the number of such therapeutics approved for clinical use is still low [1]. First of all, the reason is the nature of nucleic acids, which are macromolecules Publisher’s Note: MDPI stays neutral with a high negative charge and therefore are unable to penetrate into the cell on their with regard to jurisdictional claims in own [2]. In addition, nucleic acids are very sensitive to nucleases, as evidenced by the published maps and institutional affil- low stability of nucleic acids in physiological fluids [3]. These obstacles can be overcome iations. by (i) the development of modifications of nucleic acids and (ii) the creation of delivery systems that ensure not only penetration into the cell but also longer half-life of the nucleic acid formulation in the human body. During the past several decades, a variety of systems of nucleic acid delivery have Copyright: © 2021 by the authors. been proposed [4–8]. A distinct field of research and development of these systems is the Licensee MDPI, Basel, Switzerland. use of solid (metal) nanoparticles enclosed in a shell and serving as a carrier of a nucleic This article is an open access article acid. For instance, there are reports on the efficient delivery of small interfering RNA distributed under the terms and (siRNA) via gold nanoparticles (AuNPs) [9] and selenium nanoparticles [10] coated with conditions of the Creative Commons chitosan and gold nanorods covered with two layers of polyelectrolytes [11]. Attribution (CC BY) license (https:// Our study was aimed at creating multilayer nanoconstructs (MLNCs) based on spher- creativecommons.org/licenses/by/ ical AuNPs on whose surface an siRNA layer is adsorbed and surrounded by a lipid 4.0/). Nanomaterials 2021, 11, 2775. https://doi.org/10.3390/nano11112775 https://www.mdpi.com/journal/nanomaterials Nanomaterials 2021, 11, x 2 of 16 Nanomaterials 2021, 11, 2775 2 of 16 (siRNA) via gold nanoparticles (AuNPs) [9] and selenium nanoparticles [10] coated with chitosan and gold nanorods covered with two layers of polyelectrolytes [11]. Our study was aimed at creating multilayer nanoconstructs (MLNCs) based on spherical AuNPs on whose surface an siRNA layer is adsorbed and surrounded by a lipid envelope (Figure 1). The choice of AuNPs as the basis for this nanocomposite is due to envelope (Figure 1). The choice of AuNPs as the basis for this nanocomposite is due to their unique physicochemical properties and good biocompatibility [12–14]. their unique physicochemical properties and good biocompatibility [12–14]. Figure 1. The fabrication and structure of the desired MLNC. Figure 1. The fabrication and structure of the desired MLNC. In the present study, the fabrication of an MLNC includes several steps: preparation In the present study, the fabrication of an MLNC includes several steps: preparation of of core nanoparticles (AuNP-siRNA) and a lipid film, assembly of the MLNC, and purifi- core nanoparticles (AuNP-siRNA) and a lipid film, assembly of the MLNC, and purification cation and concentration of the nanoconstruct particles. One of the previously developed and concentration of the nanoconstruct particles. One of the previously developed versions versions of an MLNC—whose lipid envelope is doped with peptide (LR)4G conjugated of an MLNC—Whose lipid envelope is doped with peptide (LR) G conjugated with stearic with stearic acid—successfully delivers siRNA into cells and ensures a release of the acid—Successfully delivers siRNA into cells and ensures a release of the siRNA cargo, as siRNA cargo, as confirmed by the suppression of reporter protein expression (green fluo- confirmed by the suppression of reporter protein expression (green fluorescent protein; rescent protein; GFP) [15]. In that study, we showed the feasibility of siRNA delivery via GFP) [15]. In that study, we showed the feasibility of siRNA delivery via constructs constructs containing lipid-coated AuNPs [15]. On the other hand, designing formulations containing lipid-coated AuNPs [15]. On the other hand, designing formulations intended intended for clinical use requires higher nanoconstruct quality, which can be achieved by for clinical use requires higher nanoconstruct quality, which can be achieved by improving improving the method of MLNC fabrication and will enhance the efficiency of siRNA de- the method of MLNC fabrication and will enhance the efficiency of siRNA delivery into livery into human cells. human cells. The second aim of this work was to optimize the conditions of MLNC synthesis that The second aim of this work was to optimize the conditions of MLNC synthesis yield the most homogeneous suspensions free of empty lipid particles, naked core nano- that yield the most homogeneous suspensions free of empty lipid particles, naked core particles, and aggregates of core nanoparticles. To increase the efficiency of MLNC syn- nanoparticles, and aggregates of core nanoparticles. To increase the efficiency of MLNC thesis, we varied the conditions of all steps of this process on the basis of our previously synthesis, we varied the conditions of all steps of this process on the basis of our previously obt obtained ained and andpublish published ed da data ta abo about ut the st theabil stability ity and properti and properties es of nanoconstructs. In pa of nanoconstructs. In r- ticular, we varied the ratio of core nanoparticles to a lipid mixture; an optimal buffer was particular, we varied the ratio of core nanoparticles to a lipid mixture; an optimal buffer chosen t was chosen hat e that nable enables s effect efi fective ve wrapping wrapping of t of hethe core cor n eananoparticles noparticles wit with h a alip lipid id eenvelope; nvelope; and op and optimal timal c conditions onditions we wer re fo e found und for t for the he fr fractionation actionation and and p purification urification o of f t the he MLNC MLNCs. s. 2. Materials and Methods 2. Materials and Methods 2.1. Chemicals 2.1. Chemicals Tetrachloroauric acid trihydrate (HAuCl 3H O) was purchased from Aurat (Moscow, 4 2 Tetrachloroauric acid trihydrate (HAuCl4·3H2O) was purchased from Aurat (Mos- Russia), and RNA phosphoramidites for oligoribonucleotide synthesis were acquired from cow, Russia), and RNA phosphoramidites for oligoribonucleotide synthesis were ac- Sigma-Aldrich (Hamburg, Germany). Sodium chloride (NaCl) and magnesium sulfate quired from Sigma-Aldrich (Hamburg, Germany). Sodium chloride (NaCl) and magne- (MgSO ) were bought from Honeywell (Seelze, Germany) and sodium citrate dihydrate sium sulfate (MgSO4) were bought from Honeywell (Seelze, Germany) and sodium citrate (Na C H O 2H O) from Fluka (Buchs, Switzerland). Magnesium acetate tetrahydrate 3 6 5 7 2 dihydrate (Na3C6H5O7·2H2O) from Fluka (Buchs, Switzerland). Magnesium acetate tetra- [Mg(CH COO) 4H O] was acquired from VWR International LLC (Radnor, PA, USA), 3 2 2 hydrate [Mg(CH3COO)2·4H2O] was acquired from VWR International LLC (Radnor, PA, whereas egg phosphatidylcholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine USA), whereas egg phosphatidylcholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanola- (DOPE) was obtained from Avanti (Alabaster, AL, USA). 2-[[4-Dodecylamino-6-oleylamino- mine (DOPE) was obtained from Avanti (Alabaster, AL, USA). 2-[[4-Dodecylamino-6- 1,3,5-triazine-2yl]-(2-hydroxyethyl)amino]ethanol (DOME2) was synthetized as described oleylamino-1,3,5-triazine-2yl]-(2-hydroxyethyl)amino]ethanol (DOME2) was synthetized in ref. [13]. Disodium phosphate dihydrate (NaH PO 2H O) and monosodium phos- 2 4 2 as described in ref. [13]. Disodium phosphate dihydrate (NaH2PO4·2H2O) and monoso- phate dodecahydrate (Na HPO 12H O) were purchased from Reatex (Moscow, Russia) 2 4 2 dium phosphate dodecahydrate (Na2HPO4·12H2O) were purchased from Reatex (Mos- and uranyl acetate from SPI (West Chester, PA, USA). Peptide NH -(RL) G-C(O)NH 2 4 2 cow, Russia) and uranyl acetate from SPI (West Chester, PA, USA). Peptide NH2-(RL)4G- 5CF COOH was acquired from Diapharm (Lyubertsy, Russia). Sodium acetate trihydrate (CH COONa3H O), acetic acid (CH COOH), and sucrose (C H O ) were bought from 3 2 3 12 22 11 Panreac (Barcelona, Spain), whereas cesium chloride (CsCl) and glycerol (C H O ) were ob- 3 8 3 Nanomaterials 2021, 11, 2775 3 of 16 tained from Applichem (Darmstadt, Germany), and trichloromethane (chloroform, CHCl ) and methanol (CH OH) were purchased from Reachem (Moscow, Russia). AlamarBlue™ Cell Viability Reagent was acquired from Invitrogen (Waltham, MA, USA). Water was purified by means of a Simplicity 185 water purification system (Millipore, Burlington, MA, USA) and had a resistivity of 18.2 MWcm at 25 C. 2.2. Preparation of Core Nanoparticles To obtain core nanoparticles (AuNP-siRNA), siRNA was used that suppresses GFP synthesis in cultured cells stably expressing this protein. The sequence of the siRNA was 0 0 as follows: sense strand, 5 -CAAGCUGACCCUGAAGUUCTT; and antisense strand, 5 - GAACUUCAGGGUCAGCUUGTT [16]. The synthesis of this siRNA is described in detail in our previous work [17]. The AuNPs were synthesized using a previously published technique [18]. The core nanoparticles, which are AuNPs noncovalently covered with siRNA, were fabricated as described elsewhere [15,19], with some modifications: 4 mM citrate–stabilized AuNPs (size 12 1 nm according to transmission electron microscopy [TEM] or 17.3 2.1 nm according to dynamic light scattering [DLS] analysis; a zeta potential of 33.6  2.0 mV according to DLS) at a concentration of 3.6  10 M were incubated with 0.72 M siRNA for 22 h at room temperature in the presence of either 5 or 10 mM NaCl and either Mg(CH COO) or MgSO . The resultant suspensions of core nanoparticles were cen- 3 2 4 trifuged, the supernatant was discarded, and the pellet containing the core nanoparticles was washed with 1 mL of 4 mM sodium citrate dihydrate. The size and monodispersity of the obtained core nanoparticles were determined using TEM (13  1 nm) and DLS (25 9 nm). The zeta potential of the core nanoparticles proved to be44 1 mV, and the polydispersity index (PDI) of the suspension was 0.212 0.011. The core nanoparticles had a surface plasmon resonance maximum at 520 nm, and the density of siRNA molecules on the AuNP surface was 29  6. 2.3. Preparation of a Lipid Film The lipid film was prepared as described before [15], with modifications: 90 L of 1 mM egg phosphatidylcholine and DOPE in a CHCl /CH OH mixture (1:1) and 10 L 3 3 of 1 mM DOME2 in CHCl were added to 1 mL of CHCl in a 10 mL round-bottom flask. 3 3 Next, the solvent was evaporated either at 25 C and 6 mmHg or without thermostatting at 12 mmHg. The resultant lipid film was then dried in vacuum in a desiccator to remove traces of the organic solvents, after which the flask with the film was incubated at 18 C for 16 h. All the procedures for obtaining the lipid film were carried out in an argon atmosphere. 2.4. Synthesis of a Peptide Conjugate with Stearic Acid A conjugate of a peptide [(RL) G-NH ] with stearic acid [Str-(RL) G-NH ] for doping 4 2 4 2 the lipid envelope was synthesized as described previously [15]. 2.5. Assembly of the MLNC The procedure was performed as described earlier [15], with slightly modified reaction conditions, as described below. The MLNCs were obtained in two stages: First, a buffer with pH 4.5 was applied to the surface of the lipid film: either (1) 0.9 mL of H O and 31 L of NaH PO (1.00 M, 0.10 M, or 0.01 M) or (2) 0.825 mL of H O; to the 2 2 4 2 buffer, we added 100 L of a suspension of the core nanoparticles (2.5 pmol in terms of gold) in 6 mM CH COOH. The reaction mixture of the core nanoparticles and lipid film was sonicated at 90 W for 15 min at 25 C. The expected result of this step is a suspension of the core nanoparticles bearing a layer of noncovalently attached siRNA enclosed in a lipid envelope. Second stage: the pH of the suspension containing the core nanoparticles carrying the lipid envelope was adjusted to 7.4 by the addition of either (1) 69 L of 1.00, 0.10, or 0.01 M Nanomaterials 2021, 11, 2775 4 of 16 Na HPO or (2) 100 L of 3 mM CH COONa. Then, 10 L of 1 mM stearic-acid-conjugated 2 4 3 peptide, Str-(LR) G, was introduced. The reaction mixture was sonicated (90 W) for 5 min at 25 C. The size and monodispersity of the obtained MLNCs were verified using TEM and DLS. The MLNCs had a surface plasmon resonance maximum at 534 nm. Thus, we obtained a suspension of MLNCs whose envelope was doped with a peptide. For brevity, hereafter, these nanoparticles are referred to as “MLNCs,” without a mention of the peptide. 2.6. Purification of the MLNCs by Banding Centrifugation Fractionation of the MLNCs was carried out either {1} in a cesium chloride gradient or {2} in solutions of glycerol or sucrose. {1} In a 1.5 mL test tube, we sequentially layered 500 L each of an aqueous 61.7% CsCl solution ( = 1.789 g/cm ,  = 1.238 9 cP) and an aqueous 10.8% CsCl solution ( = 1.0804 g/cm ,  = 0.966 cP) and then 50 L of the MLNC suspension; its colloidal stability was examined by means of changes in the color of the MLNC layer. {2} In a 15 mL test tube, we layered 500–1000 L of the MLNC suspension on top of 10 mL of (i) an aqueous 75% glycerol solution ( = 41 cP) or (ii) an aqueous 26% sucrose solution in 1 mM phosphate buffer (pH was altered) ( = 1.1082 g/mL,  = 2.223 cP) or (iii) an aqueous 58% sucrose solution in 1 mM phosphate buffer ( = 1.267 g/mL, = 42.8 cP), followed by centrifugation at 25 C in the range of 1000–8000 g for 15–90 min. Fractions of different colors were collected into separate 1.5 mL tubes and were subjected to purification to remove excess glycerol or sucrose via dialysis or centrifugation. After that, all the fractions were analyzed using DLS and TEM. After the fractionation in sucrose, the samples with or without an added equal volume of 1 mM phosphate buffer were centrifuged in 1.5 mL tubes at 2000 g for 15, 30, or 60 min, the supernatant was discarded, and the resulting MLNCs were analyzed via DLS, TEM, and UV-Vis spectroscopy. After the fractionation in glycerol, dialysis was performed either in Centricon Plus-70 3 kDa, Merck KGaA (Darmstadt, Germany) or in screw cap 1.5 mL tubes at 25 C for 16–72 h against 1, 3, or 10 mM phosphate buffer using either (1) membranes for extruders with a pore diameter of 30 or 100 nm, Whatman (Maidstone, UK) or (2) single-layer 3.5 or 10 kDa dialysis bags, Thermo Fisher Scientific (Göteborg, Sweden). 2.7. Examination of the Composition and Quality of the Samples 2.7.1. Optical Extinction Spectra To verify colloidal stability and to determine gold concentration, we performed UV-Vis spectroscopy. Optical adsorption spectra of the core nanoparticles and all preparations of MLNCs (Figure 2) were recorded on a Clariostar plate fluorimeter, BMG Labtech (Orten- berg, Germany) in the range 400–800 nm according to the manufacturer ’s instructions. The absorption maximum of all versions of MLNC preparations was found to be shifted by 12 nm relative to AuNPs and core nanoparticles; this shift emerged after the lipid envelope formation and was not affected by other experimental conditions. Preservation of colloidal stability of the samples during the procedure was confirmed by the absence of a peak in the range 600–700 nm. Nanomaterials 2021, 11, 2775 5 of 16 Nanomaterials 2021, 11, x 5 of 16 Figure 2. “Typical” spectra of the obtained samples of AuNPs, core nanoparticles, and MLNCs. The Figure 2. “Typical” spectra of the obtained samples of AuNPs, core nanoparticles, and MLNCs. vertical lines indicate absorption maxima for AuNPs and core nanoparticles (520 nm, left-hand line) The vertical lines indicate absorption maxima for AuNPs and core nanoparticles (520 nm, left-hand and for MLNCs (532 nm, right-hand line). For clarity, we present the spectra of the samples at con- line) and for MLNCs (532 nm, right-hand line). For clarity, we present the spectra of the samples at centrations that ensure resolution of the lines in the graph. concentrations that ensure resolution of the lines in the graph. The yield of MLNCs was calculated from optical density of the final suspension at The yield of MLNCs was calculated from optical density of the final suspension 520 nm using the molar extinction coefficient characteristic of the original AuNPs (ε = 8.78 at 520 nm using the molar extinction coefficient characteristic of the original AuNPs × 10 L·mol/cm) [20]. The amount (mol) of AuNPs used to fabricate the core nanoparticles (" = 8.78  10 Lmol/cm) [20]. The amount (mol) of AuNPs used to fabricate the core was set to 100%. nanoparticles was set to 100%. 2.7.2. DLS 2.7.2. DLS All All samples sampleof s ocor f core e nanoparticles nanoparticles and and MLNCs MLNCwer s we ere c characterized haracterized via via D DLS LS and and TEM. TEM. DLS DLS ana analysis lysis is is employed employed f for or assessing assessing o overall verall hy hydr drod odynamic ynamic char characterist acteristics ics of of a suspen a sus- - pension of nanoparticles, whereas TEM provides information on the fine structure of sion of nanoparticles, whereas TEM provides information on the fine structure of nano- nanoparticles and its changes. These methods complement each other and, taken together, particles and its changes. These methods complement each other and, taken together, help help to exhaustively characterize samples of nanoconstructs. to exhaustively characterize samples of nanoconstructs. Suspensions of the core nanoparticles and MLNCs were analyzed using DLS by means Suspensions of the core nanoparticles and MLNCs were analyzed using DLS by of a Malvern Zetasizer Nano instrument, Malvern Instruments (Worcestershire, UK) in means of a Malvern Zetasizer Nano instrument, Malvern Instruments (Worcestershire, accordance with the manufacturer ’s instructions. This method allowed us to characterize UK) in accordance with the manufacturer’s instructions. This method allowed us to char- particle suspensions in terms of the following parameters: the hydrodynamic diameter, acterize particle suspensions in terms of the following parameters: the hydrodynamic di- PDI, and zeta () potential. The measurements were performed at least in triplicate. ameter, PDI, and zeta (ζ) potential. The measurements were performed at least in tripli- cate. 2.7.3. TEM For examination using TEM, 10 L of a suspension of the core nanoparticles or MLNCs 2.7.3. TEM was applied to a formvar film on a grid and incubated for 1 min. Then, the drop was For examination using TEM, 10 μL of a suspension of the core nanoparticles or aspirated with a pipettor, and without drying, the grid was placed on a drop of uranyl MLNCs was applied to a formvar film on a grid and incubated for 1 min. Then, the drop acetate for 10 s; the excess liquid was removed with filter paper. All TEM samples were was aspirated with a pipettor, and without drying, the grid was placed on a drop of uranyl prepared identically; at least five grid cells were examined in different parts of each grid. acetate for 10 s; the excess liquid was removed with filter paper. All TEM samples were The suspensions of the core nanoparticles and MLNCs were investigated under a JEM prepared identically; at least five grid cells were examined in different parts of each grid. 1400 transmission electron microscope, Jeol (Tokyo, Japan) equipped with a Veleta digital The suspensions of the core nanoparticles and MLNCs were investigated under a JEM camera, EM SIS (Muenster Germany). Particle sizes were determined using iTEM software, 1400 transmission electron microscope, Jeol (Tokyo, Japan) equipped with a Veleta digital version 5.2, EM SIS (Muenster, Germany). camera, EM SIS (Muenster Germany). Particle sizes were determined using iTEM soft- ware, version 5.2, EM SIS (Muenster, Germany). Nanomaterials 2021, 11, 2775 6 of 16 2.8. Cytotoxicity Assays of Glycerol and Sucrose To this end, SC-1 R780 fibroblasts were cultured in the IMDM medium, Gibco (Grand Island, NY, USA) supplemented with 10% of fetal calf serum (Gibco), penicillin (100 U/mL), and streptomycin (100 g/mL) in a humidified atmosphere containing 5% of CO at 37 C. The cytotoxicity assay was performed using the AlamarBlue test [21]. 2.9. Statistical Analysis Each experiment was conducted independently at least three times. Data are presented as mean  standard deviation from at least three independent experiments. 3. Results 3.1. The Mechanism of MLNC Formation The formation of a lipid envelope around the core nanoparticle is mediated by elec- trostatic interaction between protonated amino groups of the lipid (DOME2, which is a component of the lipid film) and negatively charged phosphate groups of the siRNA on the surface of the core nanoparticle. Because DOME2 is protonated at a pH below 7, the assembly of the nanoconstructs was carried out in an acidic medium. This strategy necessitated selecting a buffer that makes it easy to switch DOME2 to either a charged (protonated) state or a neutral (deprotonated) state and ensures colloidal stability of the core nanoparticles. We believe that choosing such a buffer will reduce the aggregation of MLNC particles and aggregation of “naked” core nanoparticles. To form a lipid envelope around the core nanoparticle, a suspension of the core nanoparticles in an acidic buffer was applied to the prepared lipid film, and the reaction mixture was sonicated (Figure 3A). The lipid film, which carries the core nanoparticles adsorbed on the surface, gets fragmented, and the core nanoparticles end up in a lipid envelope. The sizes of the forming particles are different, as are the numbers of core nanoparticles enclosed in a single shell. Consequently, the resulting suspension contains structurally diverse desired MLNCs (“target MLNCs”: Figure 3B, panel 1), aggregates of the core nanoparticles surrounded by a thin lipid envelope (Figure 3B, panel 2), lipid particles and empty vesicles (Figure 3B, panel 3), lipid film fragments of various sizes with adhered core nanoparticles (Figure 3B, panel 4), and naked core nanoparticles, both stand-alone and aggregated (Figure 3B, panel 5). It is obvious that the ideal end product should consist mainly of stand-alone MLNCs each containing one core nanoparticle. The presented mechanism of MLNC formation makes this outcome a pipe dream, mainly owing to the heterogeneity of adsorption of the core nanoparticles on the lipid film. Therefore, our efforts were focused on obtain- ing suspensions with the highest proportion of stand-alone MLNCs containing 1–10 core nanoparticles and a distinct electron-transparent envelope; such nanoconstructs are here- inafter referred to as “target MLNCs” (Figure 3B, panel 1). Meanwhile, we strived to reduce the concentrations of empty lipid particles, aggregates of core nanoparticles enclosed in a thin lipid envelope, and naked (unenveloped) core nanoparticles (Figure 3B, panels 2–5). It should be noted that we always assessed the effect of one or another modification of the protocol by means of changes in the characteristics of the end product: the final suspension of MLNCs. Nanomaterials 2021, 11, 2775 7 of 16 Nanomaterials 2021, 11, x 7 of 16 Figure 3. (A) The scheme of MLNC formation after the application of a suspension of the core na- Figure 3. (A) The scheme of MLNC formation after the application of a suspension of the core noparticles to the lipid film and sonication. (B) Morphology of the particles in the MLNC prepara- nanoparticles to the lipid film and sonication. (B) Morphology of the particles in the MLNC prepa- tions. Panel 1: different subtypes of a “target MLNC” containing 1 to 10 electron-dense core nano- rations. Panel 1: Different subtypes of a “target MLNC” containing 1 to 10 electron-dense core particles; Panel 2: aggregates of the core nanoparticles surrounded by a thin lipid envelope; Panel nanoparticles; Panel 2: Aggregates of the core nanoparticles surrounded by a thin lipid envelope; 3: empty lipid particles; Panel 4: fragments of the lipid film with adhered core nanoparticles; Panel Panel 5: “naked” cor 3: Empty lipid e nanoparticles. The scale particles; Panel 4: Fragments bars correspond t of the lipid o 50 nm. Nega film with adher tive edstaining with uranyl core nanoparticles; acetate followed by TEM. Panel 5: “Naked” core nanoparticles. The scale bars correspond to 50 nm. Negative staining with uranyl acetate followed by TEM. 3.2. Assembly of the Core Nanoparticles: AuNP-siRNA 3.2. Assembly of the Core Nanoparticles: AuNP-siRNA The first step in the fabrication of MLNCs is the synthesis of core nanoparticles The first step in the fabrication of MLNCs is the synthesis of core nanoparticles (AuNP- (AuNP-siRNA); we have described this procedure in detail previously [15,22,23]. For en- siRNA); we have described this procedure in detail previously [15,22,23]. For enveloping veloping the core nanoparticles with the siRNA, a suitable buffer was experimentally se- the core nanoparticles with the siRNA, a suitable buffer was experimentally selected, lected, phosphate buffer, which enabled easy switching from pH 5 to pH 7.4, which cor- phosphate buffer, which enabled easy switching from pH 5 to pH 7.4, which corresponds responds to physiological pH. It is important that this buffer ensured colloidal stability of to physiological pH. It is important that this buffer ensured colloidal stability of the core the core nanoparticles throughout the entire experiment on MLNC fabrication. nanoparticles throughout the entire experiment on MLNC fabrication. During the preparation of MLNCs, it became apparent that the lipid particles, which During the preparation of MLNCs, it became apparent that the lipid particles, which contained lipid DOME2, had negative ζ potential in acidic phosphate buffer, indicating contained lipid DOME2, had negative  potential in acidic phosphate buffer, indicating adsorption of phosphate anions on the surface of the lipid film. Accordingly, the phos- adsorption of phosphate anions on the surface of the lipid film. Accordingly, the phosphate phate component of the buffer will compete with the core nanoparticles during their ad- component of the buffer will compete with the core nanoparticles during their adsorption sorption onto the lipid film, thereby inevitably decreasing the efficiency of the enveloping onto the lipid film, thereby inevitably decreasing the efficiency of the enveloping process. process. At first glance, the simplest solution seems to be to change the buffer, but this At first glance, the simplest solution seems to be to change the buffer, but this approach approach can diminish colloidal stability of the core nanoparticles. We reduced the phos- can diminish colloidal stability of the core nanoparticles. We reduced the phosphate anion phate anion concentration from 0.100 to 0.001 M, which increased the efficiency of MLNC concentration from 0.100 to 0.001 M, which increased the efficiency of MLNC formation formation (Figure 4) and decreased the PDI (Table 1). (Figure 4) and decreased the PDI (Table 1). Nanomaterials 2021, 11, x 8 of 16 Table 1. Physicochemical parameters of MLNCs at different concentrations of phosphate buffer (PB). MLNC Sample ID PB Concentration, mM PDI Hydrodynamic Diameter, nm 1 100 0.203 ± 0.012 749.2 ± 177 Nanomaterials 2021, 11, 2775 8 of 16 2 10 0.256 ± 0.010 169 ± 88 3 3 0.275 ± 0.015 137.7 ± 65 4 1 0.209 ± 0.009 158.7 ± 74 Figure 4. Ultrastructure of the obtained MLNCs. (A) Assembly in 100 mM phosphate buffer, (B) assembly in 10 mM phos- Figure 4. Ultrastructure of the obtained MLNCs. (A) Assembly in 100 mM phosphate buffer, (B) assembly in 10 mM phate buffer, and (C) assembly in 1 mM phosphate buffer. The scale bars correspond to 100 nm. Negative staining with a phosphate buffer, and (C) assembly in 1 mM phosphate buffer. The scale bars correspond to 100 nm. Negative staining with 0.5% uranyl acetate solution followed by TEM. a 0.5% uranyl acetate solution followed by TEM. By lowering the buffer concentration, we reduced the amount of phosphate anions Table 1. Physicochemical parameters of MLNCs at different concentrations of phosphate buffer (PB). adsorbed on the lipid film surface; however, it was necessary to neutralize the effect of the remaining ones. For this purpose, it was decided to add a divalent cation that would act MLNC Sample ID PB Concentration, mM PDI Hydrodynamic Diameter, nm as a linker between the phosphate anions adsorbed on the lipid film surface and the siRNA 1 2+100 0.203 0.012 749.2  177 on the surface of AuNPs. Mg was chosen and did not lead to “adhesion” and precipita- 2 10 0.256 0.010 169  88 tion of the components of the lipid film and core nanoparticles, nor does it catalyze phos- 3 3 0.275 0.015 137.7  65 phodiester bond hydrolysis in siRNA. Nevertheless, which salt can be used in this con- 4 1 0.209 0.009 158.7  74 text? It is known that the type of anion has a pronounced effect on colloidal stability of core nanoparticles [24–26]. Accordingly, we introduced either magnesium acetate or mag- By lowering the buffer concentration, we reduced the amount of phosphate anions nesium sulfate into the reaction mixture. adsorbed on the lipid film surface; however, it was necessary to neutralize the effect of During the synthesis of the core nanoparticles, the influence of Mg(CH3COO)2 or the remaining ones. For this purpose, it was decided to add a divalent cation that would MgSO4 at 0.1 or 0.4 mM added to the reaction mixture was determined by means of phys- act as a linker between the phosphate anions adsorbed on the lipid film surface and the icochemical characteristics (Table 2) and alterations of colloidal stability of the core nano- 2+ siRNA on the surface of AuNPs. Mg was chosen and did not lead to “adhesion” and particle suspension. Acetate ions at both concentrations affected the colloidal stability neg- precipitation of the components of the lipid film and core nanoparticles, nor does it catalyze atively. The addition of magnesium ions in the form of 0.1 mM sulfate “improved” hy- phosphodiester bond hydrolysis in siRNA. Nevertheless, which salt can be used in this drodynamic parameters of the core nanoparticles and did not alter colloidal stability for context? It is known that the type of anion has a pronounced effect on colloidal stability several days. At 0.4 mM, the impact of this salt was negative. of core nanoparticles [24–26]. Accordingly, we introduced either magnesium acetate or magnesium sulfate into the reaction mixture. Table 2. Physicochemical characteristics of the core nanoparticles in phosphate buffer containing different magnesium During the synthesis of the core nanoparticles, the influence of Mg(CH COO) or 3 2 salts. MgSO at 0.1 or 0.4 mM added to the reaction mixture was determined by means of Sample Magnesium Salt ζ Potential, mV Hydrodynamic Diameter, nm PDI physicochemical characteristics (Table 2) and alterations of colloidal stability of the core Core nanoparticles nanoparticle none suspension.−Acetate 44 ± 1 ions at both concentrations 25 ± 9 affected the colloidal 0.212 ± 0. stability 011 negatively. The addition of magnesium ions in the form of 0.1 mM sulfate “improved” Core nanoparticles 0.1 mM Mg(Ac)2 −45 ± 1 27 ± 11 0.416 ± 0.041 hydrodynamic parameters of the core nanoparticles and did not alter colloidal stability for Core nanoparticles 0.1 mM MgSO4 −39 ± 1 25 ± 9 0.266 ± 0.050 several days. At 0.4 mM, the impact of this salt was negative. Core nanoparticles 0.4 mM MgSO4 −40 ± 2 90 ± 85 0.565 ± 0.316 Table 2. Physicochemical characteristics of the core nanoparticles in phosphate buffer containing different magnesium salts. Having found the optimal concentration of MgSO4 (0.1 mM) for obtaining the core nanoparticles, we began to vary the concentration of NaCl, the presence of which is nec- Sample Magnesium Salt  Potential, mV Hydrodynamic Diameter, nm PDI essary for denser loading of siRNA molecules onto AuNPs. AuNPs are very sensitive to Core nanoparticles none 44  1 25  9 0.212  0.011 Core nanoparticles 0.1 mM Mg(Ac) 45  1 27  11 0.416  0.041 Core nanoparticles 0.1 mM MgSO 39  1 25  9 0.266  0.050 Core nanoparticles 0.4 mM MgSO 40  2 90  85 0.565  0.316 Having found the optimal concentration of MgSO (0.1 mM) for obtaining the core nanoparticles, we began to vary the concentration of NaCl, the presence of which is nec- Nanomaterials 2021, 11, 2775 9 of 16 Nanomaterials 2021, 11, x 9 of 16 essary for denser loading of siRNA molecules onto AuNPs. AuNPs are very sensitive to monovalent anions [25], and even a small shift in their concentration can affect colloidal monovalent anions [25], and even a small shift in their concentration can affect colloidal stability of the resulting core nanoparticles. Previously [15], we prepared core nanoparti- stability of the resulting core nanoparticles. Previously [15], we prepared core nanoparti- cles at 10 mM NaCl (parameters of the obtained preparations of MLNCs: hydrodynamic cles at 10 mM NaCl (parameters of the obtained preparations of MLNCs: hydrodynamic diameter of 205  100 nm and PDI of 0.186), but in the present work, we reduced the diameter of 205 ± 100 nm and PDI of 0.186), but in the present work, we reduced the NaCl NaCl concentration in half, which increased suspension homogeneity and reduced the concentration in half, which increased suspension homogeneity and reduced the size of size of MLNCs (hydrodynamic diameter became 128  54 nm, and PDI became 0.151). MLNCs (hydrodynamic diameter became 128 ± 54 nm, and PDI became 0.151). The change The change in NaCl concentration did not lead to a noticeable alteration of the end prod- in NaCl concentration did not lead to a noticeable alteration of the end products’ compo- ucts’ composition according to TEM; they contained MLNCs with different numbers of sition according to TEM; they contained MLNCs with different numbers of core nanopar- core nanoparticles as well as lipid particles, empty lipid vesicles, aggregates of the core ticles as well as lipid particles, empty lipid vesicles, aggregates of the core nanoparticles, nanoparticles, and a small percentage of naked core nanoparticles (Figure 5). In subsequent and a small percentage of naked core nanoparticles (Figure 5). In subsequent experiments, experiments, the reaction mixture version containing 0.1 mM MgSO and 5 mM NaCl was the reaction mixture version containing 0.1 mM MgSO4 and 5 mM NaCl was utilized to utilized to synthesize the core nanoparticles. synthesize the core nanoparticles. Figure 5. Ultrastructure of the MLNCs obtained in the presence of 0.1 mM MgSO4. (A) MLNCs Figure 5. Ultrastructure of the MLNCs obtained in the presence of 0.1 mM MgSO . (A) MLNCs prepared via the addition of 10 mM NaCl or (B) 5 mM NaCl. The scale bars correspond to 200 nm. prepared via the addition of 10 mM NaCl or (B) 5 mM NaCl. The scale bars correspond to 200 nm. Negative staining with a 0.5% uranyl acetate solution followed by TEM. Negative staining with a 0.5% uranyl acetate solution followed by TEM. 3.3. Fabrication of the Lipid Film 3.3. Fabrication of the Lipid Film The lipid film was generated via evaporation of a solution of mixed lipids in a chlo- The lipid film was generated via evaporation of a solution of mixed lipids in a chloro- roform/methanol mixture under reduced pressure (6 mmHg). We found that the evapo- form/methanol mixture under reduced pressure (6 mmHg). We found that the evaporation ration rate, which can be adjusted by changing pressure, affects the quality of the resulting rate, which can be adjusted by changing pressure, affects the quality of the resulting film: an film: an increase in pressure from 6 to 12 mmHg and elimination of flask thermostatting increase in pressure from 6 to 12 mmHg and elimination of flask thermostatting improved improved the homogeneity of the lipid film and facilitated its interaction with the core the homogeneity of the lipid film and facilitated its interaction with the core nanoparticles. nanoparticles. Accordingly, more homogeneous end products with a higher concentration Accordingly, more homogeneous end products with a higher concentration of MLNCs were of MLNCs were obtained. For instance, the hydrodynamic diameter of MLNCs decreased obtained. For instance, the hydrodynamic diameter of MLNCs decreased from 188  96 to from 144.3 18 8 ± 9 79 nm, 6 to 1 while 44.3 ± the 79 nm PDI, was whil0.259 e the  PDI 0.005 was 0. nm2and 59 ± 0. 0.250 005 and  0.006 0.25 nm, 0 ± 0. respectively 006, respec- . TEM tively. TE analysis M analy of these sis of the sample se samp s revealed les reno veale app d no ap reciable preciab differ le differences in the ences in their composition: ir compo- they contained MLNCs of various structures, empty lipid particles, aggregates of the core sition: they contained MLNCs of various structures, empty lipid particles, aggregates of nanoparticles the core nano within particle as w thin ith lipid in a t envelope, hin lipid envelope, and naked an cor d nak e nanoparticles ed core nanopart (Figur ic ele 6s ). (Figure 6). Nanomaterials 2021, 11, 2775 10 of 16 Nanomaterials 2021, 11, x 10 of 16 Figure 6. Ultrastructure of the MLNCs prepared from the lipid film synthesized under a pressure Figure 6. Ultrastructure of the MLNCs prepared from the lipid film synthesized under a pressure of of 12 mmHg (A) or 6 mmHg (B). The scale bars correspond to 200 nm. Negative staining with a 0.5% 12 mmHg (A) or 6 mmHg (B). The scale bars correspond to 200 nm. Negative staining with a 0.5% uranyl acetate solution followed by TEM. uranyl acetate solution followed by TEM. 3.4. Doping of the Lipid Envelope of MLNCs with the Peptide 3.4. Doping of the Lipid Envelope of MLNCs with the Peptide The final step in the fabrication of the target MLNCs is doping of the lipid envelope The final step in the fabrication of the target MLNCs is doping of the lipid envelope with peptide Str-(LR)4G at pH 7.4 to make sure that the nanoparticles can penetrate into with peptide Str-(LR) G at pH 7.4 to make sure that the nanoparticles can penetrate into the cell and to overcome endosomal arrest as well as to enable the release of the siRNA the cell and to overcome endosomal arrest as well as to enable the release of the siRNA into into the cytosol from the surface of the core nanoparticle. The effectiveness of this proce- the cytosol from the surface of the core nanoparticle. The effectiveness of this procedure dure has been demonstrated by us previously [15]. In the present work, a sample of lipid- has been demonstrated by us previously [15]. In the present work, a sample of lipid-coated coated core nanoparticles obtained at pH 4.5 via sonication was transferred to a medium core nanoparticles obtained at pH 4.5 via sonication was transferred to a medium with pH with pH 7.5, after which the peptide was added, and the mixture was sonicated for 5 min 7.5, after which the peptide was added, and the mixture was sonicated for 5 min at 25 C. at 25 °C. It turned out that the doping of the lipid envelope with Str-(LR) G affects physic- It turned out that the doping of the lipid envelope with Str-(LR)4G affects physico- ochemical parameters of the resultant MLNCs: the addition of the peptide yielded an chemical parameters of the resultant MLNCs: the addition of the peptide yielded an in- increase in the hydrodynamic diameter from 118.4  44.27 to 235.1  100.3 nm, while the crease in the hydrodynamic diameter from 118.4 ± 44.27 to 235.1 ± 100.3 nm, while the PDI PDI was 0.1942 and 0.1854 for the MLNCs without doping and after the doping, respec- was 0.1942 and 0.1854 for the MLNCs without doping and after the doping, respectively. tively. Structural analysis of the end product using TEM did not uncover any noticeable Structural analysis of the end product using TEM did not uncover any noticeable changes changes in its composition and in particle structure of the MLNCs after the doping with in its composition and in particle structure of the MLNCs after the doping with peptide peptide Str-(LR) G. Str-(LR)4G. These findings allowed us to find optimal modifications of the initial protocol [15] that affor These f d theindin bestgs quality allowed of MLNCs: us to find the opt pr imal mod esence ofif0.1 icatmM ions of t MgSO he in and itial prot 5 mMoco NaCl l [15] during that affo therd t assembly he best qu of the alitcor y of MLNC e nanoparticles, s: the prlipid esence o filmf 0. synthesis 1 mM M at gSO 12 4mmHg and 5 m without M NaCl thermostatting, and the assembly of MLNCs in 1 mM phosphate buffer. The end product during the assembly of the core nanoparticles, lipid film synthesis at 12 mmHg without obtained thermostatting, under these and the conditions assembly o is herf einafter MLNCs referr in 1 mM ed to phosphate b as “optimized uffer. The en MLNCs” (Figur d product e 7). The preparations of optimized MLNCs are characterized by a hydrodynamic diameter of obtained under these conditions is hereinafter referred to as “optimized MLNCs” (Figure 152  75 nm and a PDI of 0.201  0.012. 7). The preparations of optimized MLNCs are characterized by a hydrodynamic diameter The presented modifications of the protocol of MLNC fabrication increased the ef- of 152 ± 75 nm and a PDI of 0.201 ± 0.012. ficiency of synthesis of core nanoparticles carrying the lipid envelope, but the obtained The presented modifications of the protocol of MLNC fabrication increased the effi- suspensions still contained a noticeable amount of lipid particles and vesicles, aggregates ciency of synthesis of core nanoparticles carrying the lipid envelope, but the obtained sus- of the core nanoparticles within a thin lipid envelope, and naked core nanoparticles, thus pensions still contained a noticeable amount of lipid particles and vesicles, aggregates of necessitating purification of the MLNC preparations. Given that the MLNCs, lipid parti- the core nanoparticles within a thin lipid envelope, and naked core nanoparticles, thus cles, and core nanoparticles have different densities and sizes, they can be separated using necessitating purification of the MLNC preparations. Given that the MLNCs, lipid parti- banding high-speed centrifugation. cles, and core nanoparticles have different densities and sizes, they can be separated using banding high-speed centrifugation. Nanomaterials 2021, 11, 2775 11 of 16 Nanomaterials 2021, 11, x 11 of 16 Figure 7. Ultrastructure of optimized MLNCs. The scale bar corresponds to 200 nm. Negative stain- Figure 7. Ultrastructure of optimized MLNCs. The scale bar corresponds to 200 nm. Negative ing with a 0.5% uranyl acetate solution followed by TEM. staining with a 0.5% uranyl acetate solution followed by TEM. 3.5. Fractionation of MLNCs via Centrifugation 3.5. Fractionation of MLNCs via Centrifugation Cesium chloride density gradient fractionation is the best-studied and widely used Cesium chloride density gradient fractionation is the best-studied and widely used technique for the separation of macromolecules. We fractionated optimized MLNCs in a technique for the separation of macromolecules. We fractionated optimized MLNCs in a stepwise CsCl gradient; as a consequence, the color of the sample turned from dark red to stepwise CsCl gradient; as a consequence, the color of the sample turned from dark red blue, indicating complete loss of colloidal stability of these nanoparticles in the cesium to blue, indicating complete loss of colloidal stability of these nanoparticles in the cesium chloride solution. chloride solution. After giving up on cesium chloride, for the fractionation of MLNCs, we chose an After giving up on cesium chloride, for the fractionation of MLNCs, we chose an aqueous solution of sucrose as a viscous and less “aggressive” medium. The fractionation aqueous solution of sucrose as a viscous and less “aggressive” medium. The fractionation was conducted in a homogeneous solution without a concentration gradient. The sucrose was conducted in a homogeneous solution without a concentration gradient. The sucrose concentration was selected according to calculations suggesting that target MLNCs concentration was selected according to calculations suggesting that target MLNCs should should penetrate into the viscous medium to 2 cm depth. The computation was performed penetrate into the viscous medium to 2 cm depth. The computation was performed using using a calculator [27]. To this end, the density of the nanoparticles was assumed to equal a calculator [27]. To this end, the density of the nanoparticles was assumed to equal that of gold, 19 g/cm , and we hypothesized that during the centrifugation, the less dense that of gold, 19 g/cm , and we hypothesized that during the centrifugation, the less envelope and the heavy dense core would move at different acceleration rates. In accord- dense envelope and the heavy dense core would move at different acceleration rates. In ance with the calculation results, a 58% sucrose solution was selected (ρ = 1.267 g/mL, μ = accordance with the calculation results, a 58% sucrose solution was selected ( = 1.267 g/mL, 42.8 cP). = 42.8 cP). On top of the sucrose solution, 500–1000 μL of the optimized MLNC suspension was On top of the sucrose solution, 500–1000 L of the optimized MLNC suspension layered, followed by centrifugation at 25 °C and 2000× g for 1 h. The resultant fractions was layered, followed by centrifugation at 25 C and 2000 g for 1 h. The resultant fractions were carefully collected w were carefully collected ith a dispenser. The h with a dispenser igh . The concentration high concentration of sucrosof e m sucr akeose s the makes collect the ed collected fractions unsu fractions itable for exam unsuitable for inat examination ion using TEM and acc using TEMo and rding accor ly for dingly assess for ing assessing their quality. To remove the excess sucrose, an equal volume of 1 mM phosphate their quality. To remove the excess sucrose, an equal volume of 1 mM phosphate buffer buf wa fer s a was dded added to the sa to the mp samples les of selected f of selected racti fractions ons of ML ofNCs; MLNCs; the res theur lting esulting suspens suspensions ions were wer cent e centrifuged rifuged for 1 for 5 m 15 in min at 20 at 00× 2000 g. The  g. supern The supernatant atant was d was iscarde discar d, the pellet was re ded, the pellet was sus- resuspended in 1 mM phosphate buffer, and this sample was analyzed via TEM and DLS. pended in 1 mM phosphate buffer, and this sample was analyzed via TEM and DLS. The TEM analysis of the fractions obtained via centrifugation in the 58% sucrose The TEM analysis of the fractions obtained via centrifugation in the 58% sucrose so- solution showed that the middle and upper fractions were very similar because target lution showed that the middle and upper fractions were very similar because target MLNCs constituted the bulk of each sample (Figure 8A,B). By contrast, the bottom fraction MLNCs constituted the bulk of each sample (Figure 8A,B). By contrast, the bottom fraction mainly consisted of large aggregates of the core nanoparticles enclosed in a thin lipid mainly consisted of large aggregates of the core nanoparticles enclosed in a thin lipid en- envelope and aggregates of naked core nanoparticles (Figure 8C). velope and aggregates of naked core nanoparticles (Figure 8C). Nanomaterials 2021, 11, 2775 12 of 16 Nanomaterials 2021, 11, x 12 of 16 Figure 8. Ultrastructure of the fractions of optimized MLNCs obtained via centrifugation in a 58% sucrose solution for 1 Figure 8. Ultrastructure of the fractions of optimized MLNCs obtained via centrifugation in a 58% sucrose solution for 1 h h at 25 °C and 2000× g. (A) Upper fraction, (B) middle fraction, and (C) bottom fraction. The scale bars correspond to 200 at 25 C and 2000 g. (A) Upper fraction, (B) middle fraction, and (C) bottom fraction. The scale bars correspond to 200 nm. nm. Negative staining with a 0.5% uranyl acetate solution followed by TEM. Negative staining with a 0.5% uranyl acetate solution followed by TEM. We evaluated the yield of target MLNCs after all the steps of fabrication and purifi- We evaluated the yield of target MLNCs after all the steps of fabrication and purifi- cation. For this purpose, the gold content of the target fraction was calculated from its cation. For this purpose, the gold content of the target fraction was calculated from its optical density and then divided by the amount of gold in the AuNPs used for the syn- optical density and then divided by the amount of gold in the AuNPs used for the synthesis thesis of the core nanoparticles. The yield of MLNCs (in terms of gold) in the middle frac- of the core nanoparticles. The yield of MLNCs (in terms of gold) in the middle fraction tion (Figure 8B) was 15%; the sample after the removal of excess sucrose contained 5.5% (Figure 8B) was 15%; the sample after the removal of excess sucrose contained 5.5% of of sucrose, and the hydrodynamic diameter of particles in this fraction was 195 ± 78 nm sucrose, and the hydrodynamic diameter of particles in this fraction was 195  78 nm with with a PDI of 0.173. a PDI of 0.173. The low yield of MLNCs may be related both to the low efficiency of coating of the The low yield of MLNCs may be related both to the low efficiency of coating of core nanoparticles during the fabrication of MLNCs and to degradation of the MLNC the core nanoparticles during the fabrication of MLNCs and to degradation of the MLNC sample during the fractionation because the heavy gold core can “pierce” the lipid enve- sample during the fractionation because the heavy gold core can “pierce” the lipid envelope lope during the centrifugation. Consequently, centrifugation duration was reduced to 30 during the centrifugation. Consequently, centrifugation duration was reduced to 30 min, min, which yielded four fractions represented by colored discrete rings. Characteristics of which yielded four fractions represented by colored discrete rings. Characteristics of these these fractions are given in Table 3. TEM analysis of the fractions did not reveal any ap- fractions are given in Table 3. TEM analysis of the fractions did not reveal any appreciable preciable differences in the composition and structure of their nanoparticles as compared differences in the composition and structure of their nanoparticles as compared with the with the MLNC preparations after 1 h centrifugation. MLNC preparations after 1 h centrifugation. Table 3. Physicochemical characteristics of optimized MLNCs after fractionation at 2000× g for 30 Table 3. Physicochemical characteristics of optimized MLNCs after fractionation at 2000 g min. for 30 min. Sample Hydrodynamic Diameter, nm PDI Gold Content, % Sample Hydrodynamic Diameter, nm PDI Gold Content, % Optimized MLNCs 152 ± 75 0.205 ± 0.008 - Optimized MLNCs 152  75 0.205  0.008 - Upper fraction 215 ± 108 0.198 ± 0.011 12 Upper fraction 215  108 0.198  0.011 12 Middle fraction 397 ± 163 0.160 ± 0.005 32 Middle fraction 397  163 0.160  0.005 32 Bot Bottom tom fract fraction ion 12 1259 59 ± 46  461 1 0.09 0.097 7 ± 0. 0 0.009 09 24 24 Total gold content 68 Total gold content 68 The total gold content of the four fractions was 68% of the initial gold amount in The total gold content of the four fractions was 68% of the initial gold amount in A AuNPs uNPs (u (utilized tilized f for or the sy the synthesis nthesis of core of core na nanoparticles). noparticles). One- One-thir third of d of the g the gold old (a (appar pparentl ently y in the form of naked core nanoparticles released during the disintegration of MLNCs) was in the form of naked core nanoparticles released during the disintegration of MLNCs) was di distributed stributed throughout the throughout the rest rest ((10 10 mL) mL) of of th the e sucrose so sucrose solution lution vo volume lume in in the ce the centrifuge ntrifuge tube a tube and nd was not detecta was not detectable ble by di by dir rect ob ect observation. servation. IIn n s sear earch ch of of opti optimal mal f fractionation ractionationduration, durationwe , we r reduce educe d d the the centri centri fugation fugation du duration ration to 15 min and noticed that this time enables MLNCs’ separation into two discrete fractions. to 15 min and noticed that this time enables MLNCs’ separation into two discrete frac- t The ions. The upper up fraction per fract contained ion cont13–15% ained 13 of –15% MLNCs, of MLNCs, wh which corrich corr espondsesponds to the upper to the upper fraction with the half-hour centrifugation. At the same time, an increase in the concentration of fraction with the half-hour centrifugation. At the same time, an increase in the concentra- tion of MLNCs up to 40% was observed in the middle fraction. It is this fraction (Figure 9) that we are currently testing in cell culture assays. Nanomaterials 2021, 11, 2775 13 of 16 MLNCs up to 40% was observed in the middle fraction. It is this fraction (Figure 9) that we Nanomaterials 2021, 11, x 13 of 16 are currently testing in cell culture assays. Figure 9. Ultrastructure of optimized MLNCs after 15 min fractionation in 58% sucrose and removal Figure 9. Ultrastructure of optimized MLNCs after 15 min fractionation in 58% sucrose and removal of excess sucrose. The scale bars correspond to 100 nm. Negative staining with a 0.5% uranyl acetate of excess sucrose. The scale bars correspond to 100 nm. Negative staining with a 0.5% uranyl acetate solution followed by TEM. solution followed by TEM. Our study indicates that the amount of target MLNCs in the final suspension is de- Our study indicates that the amount of target MLNCs in the final suspension is deter- termined not only by the efficiency of coating of the core nanoparticles with the lipid en- mined not only by the efficiency of coating of the core nanoparticles with the lipid envelope velope but also by MLNC preservation during the purification and concentration proce- but also by MLNC preservation during the purification and concentration procedures. We dures. We believe that the approaches that we used to improve the quality of lipid-coated believe that the approaches that we used to improve the quality of lipid-coated nanocon- nanoconstructs and the newly developed methodology will be useful to researchers cre- structs and the newly developed methodology will be useful to researchers creating similar ating similar nanoconstructs. nanoconstructs. Undoubtedly, an important factor in the proposed method is the choice of a medium Undoubtedly, an important factor in the proposed method is the choice of a medium for separating a suspension of MLNCs into fractions. In addition to sucrose and cesium for separating a suspension of MLNCs into fractions. In addition to sucrose and cesium chloride, we tested another type of viscous medium: aqueous solutions of glycerin (details chloride, we tested another type of viscous medium: aqueous solutions of glycerin (details are given in the Materials and Methods section). are given in the Materials and Methods section). The best results were obtained with the following parameters of the procedure: cen- The best results were obtained with the following parameters of the procedure: cen- trifugation in a 75% glycerol solution for 40 min at 8000× g and 25 °C with subsequent trifugation in a 75% glycerol solution for 40 min at 8000 g and 25 C with subsequent dialysis for 16 h (using membranes for extruders, pore diameter 30 nm). As in the case of dialysis for 16 h (using membranes for extruders, pore diameter 30 nm). As in the case of sucrose, in glycerol solutions, optimized MLNCs get separated into fractions (Figure 10A). sucrose, in glycerol solutions, optimized MLNCs get separated into fractions (Figure 10A). According to TEM (Figure 10B), the upper fraction contained lipid particles and vesicles According to TEM (Figure 10B), the upper fraction contained lipid particles and vesi- (hydrodynamic diameter 123 ± 55 nm, PDI = 0.366), the middle dark-red fraction contained cles (hydrodynamic diameter 123  55 nm, PDI = 0.366), the middle dark-red fraction MLNCs (hydrodynamic diameter 127 ± 40 nm, PDI = 0.279), and the bottom fraction con- contained MLNCs (hydrodynamic diameter 127  40 nm, PDI = 0.279), and the bottom sisted of large aggregates of the core nanoparticles enclosed in an “incomplete” lipid en- fraction consisted of large aggregates of the core nanoparticles enclosed in an “incomplete” velope and aggregates of core nanoparticles stuck to fragments of lipid envelopes (hydro- lipid envelope and aggregates of core nanoparticles stuck to fragments of lipid envelopes dynamic diameter 233 ± 74 nm, PDI = 0.280). (hydrodynamic diameter 233  74 nm, PDI = 0.280). We fine-tuned all stages of the fractionation of optimized MLNCs in glycerol solu- We fine-tuned all stages of the fractionation of optimized MLNCs in glycerol solutions, tions, subsequent purification via dialysis, and the concentration procedure (data not subsequent purification via dialysis, and the concentration procedure (data not shown). shown). The highest-quality end products were homogeneous, had a PDI of ~0.160, and The highest-quality end products were homogeneous, had a PDI of ~0.160, and contained contained ≥22–27% of glycerol as well as nanoparticles with a hydrodynamic diameter of 22–27% of glycerol as well as nanoparticles with a hydrodynamic diameter of 115 49 nm. 115 ± 49 nm. There were concerns that the presence of glycerol could negatively affect cell There were concerns that the presence of glycerol could negatively affect cell viability, and viability, and we performed an assay of its cytotoxicity on cultured SC-1 R780 fibroblasts. we performed an assay of its cytotoxicity on cultured SC-1 R780 fibroblasts. It turned out that when a 75% solution of glycerol is diluted 32-fold (down to 2.25% It turned out that when a 75% solution of glycerol is diluted 32-fold (down to 2.25% concentration)—which corresponds to its calculated concentration when the final MLNC concentration)—Which corresponds to its calculated concentration when the final MLNC suspension is added to the cell culture—a negative effect on cell viability is detectable. By suspension is added to the cell culture—A negative effect on cell viability is detectable. By contra contrast, st, sucrose soluti sucrose solutions ons di did d not ma not manifest nifest pronounced toxi pronounced toxicity city towa towar rd the cul d the cultur tured edfi- fibr broblasts oblasts at t at the he correspon corresponding ding di dilutions lutions (Figur (Figure 1 e 111 ). ). Nanomaterials 2021, 11, 2775 14 of 16 Nanomaterials 2021, 11, x 14 of 16 Nanomaterials 2021, 11, x 14 of 16 Figure 10. Top row: purification of optimized MLNCs via centrifugation in 75% glycerin. (A) The Figure 10. Top row: purification of optimized MLNCs via centrifugation in 75% glycerin. (A) The Figure 10. Top row: purification of optimized MLNCs via centrifugation in 75% glycerin. (A) The sample is applied to a glycerin layer; (B) separation of the sample into three fractions after centrifu- sample is applied to a glycerin layer; (B) separation of the sample into three fractions after cen- sample is applied to a glycerin layer; (B) separation of the sample into three fractions after centrifu- gation (1: upper fraction, 2: middle (target) fraction, and 3: bottom fraction). Bottom row: fractions trifugation (1: upper fraction, 2: middle (target) fraction, and 3: bottom fraction). Bottom row: gation (1: upper fraction, 2: middle (target) fraction, and 3: bottom fraction). Bottom row: fractions of optimized MLNCs obtained via centrifugation in 75% glycerol and purified via dialysis: (C) up- fractions of optimized MLNCs obtained via centrifugation in 75% glycerol and purified via dialysis: per fraction, ( of optimized MLNCs obtain D) middle fraction, and ( ed via centrifugation E) bottom fraction. The sca in 75% glycerol and p le bars correspond to urified via dialysis: ( 100 nm. C) up- (C) upper fraction, (D) middle fraction, and (E) bottom fraction. The scale bars correspond to 100 nm. per fraction, (D) middle fraction, and (E) bottom fraction. The scale bars correspond to 100 nm. Contrasting by means of a 0.5% uranyl acetate solution followed by TEM. Contrasting by means of a 0.5% uranyl acetate solution followed by TEM. Contrasting by means of a 0.5% uranyl acetate solution followed by TEM. Figure 11. Viability of SC-1 R780 fibroblasts in the presence of glycerol or sucrose. The vertical axis Figure 11. Viability of SC-1 R780 fibroblasts in the presence of glycerol or sucrose. The vertical axis denotes the percentage of viable cells, and the horizontal axis shows fold dilution of stock solutions Figure 11. Viability of SC-1 R780 fibroblasts in the presence of glycerol or sucrose. The vertical axis denotes the percentage of viable cells, and the horizontal axis shows fold dilution of stock solutions of glycerol (75%) and sucrose (58%). denotes the percentage of viable cells, and the horizontal axis shows fold dilution of stock solutions of glycerol (75%) and sucrose (58%). of glycerol (75%) and sucrose (58%). These findings indicate that the presence of glycerol in the culture medium, even at These findings indicate that the presence of glycerol in the culture medium, even at low concentrations, has an adverse effect on cells. Therefore, the MLNC preparations ob- These findings indicate that the presence of glycerol in the culture medium, even low concentrations, has an adverse effect on cells. Therefore, the MLNC preparations ob- tained using the purification method involving fractionation of samples in glycerol is not at low concentrations, has an adverse effect on cells. Therefore, the MLNC preparations tained using the purification method involving fractionation of samples in glycerol is not suitable for research on cultured cells. We present the results of this study to draw the obtained using the purification method involving fractionation of samples in glycerol is suitable for research on cultured cells. We present the results of this study to draw the Nanomaterials 2021, 11, 2775 15 of 16 not suitable for research on cultured cells. We present the results of this study to draw the readers’ attention to the necessity of comprehensive characterization before the approval of methods intended for nanobiotechnology and nanomedicine. 4. Conclusions Lipid-coated particles that serve as a carrier of siRNA are the subject of numerous studies. Several years ago, we published the proof of principle for the construction of an AuNP-based MLNC that efficiently delivers siRNA into the cell [15]. Nonetheless, we were not satisfied with the quality of the obtained nanocomposites, and thus, here we found some ways to improve it in comparison with the original version. In this work, we demonstrated that even seemingly insignificant modifications of the steps of the MLNC fabrication affect end product quality. For example, the optimal reaction mixture for obtaining the core nanoparticles contains 0.1 mM MgSO and 5 mM NaCl; lipid film synthesis at 12 mmHg without thermostatting improves the quality of the forming MLNCs, as does the assembly of MLNCs in 1 mM phosphate buffer. Having optimized all the steps of MLNC fabrication, we noticed that 15 min centrifugation at 2000 g in 58% sucrose yields a fraction containing 40% of target MLNCs, i.e., a doubled proportion of these nanoconstructs as compared to the end product of the original procedure [15]. We think that this study can help researchers who design nanoconstructs based on metal nanoparticles coated with a lipid envelope. Author Contributions: Conceptualization, I.A.P., I.S.D. and A.V.E.; methodology, I.S.D.; investi- gation, A.V.E., J.E.P., I.S.D. and B.P.C.; resources, D.V.P.; data curation, I.A.P.; writing—Original draft preparation, A.V.E., I.S.D. and I.A.P.; writing—Review and editing, E.I.R.; visualization, J.E.P.; supervision, D.V.P.; project administration, E.I.R.; funding acquisition, E.I.R. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Russian Science Foundation, grant # 19-15-00217; the synthesis of siRNA was funded by Russian State Funded Project # 121031300042-1. Data Availability Statement: Data are available on request from the corresponding author. Conflicts of Interest: The authors declare no conflict of interest. References 1. Kulkarni, J.A.; Witzigmann, D.; Thomson, S.B.; Chen, S.; Leavitt, B.R.; Cullis, P.R.; van der Meel, R. The current landscape of nucleic acid therapeutics. Nat. 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Enhanced siRNA Delivery and Selective Apoptosis Induction in H1299 Cancer Cells by Layer-by- Layer-Assembled Se Nanocomplexes: Toward More Efficient Cancer Therapy. Front. Mol. Biosci. 2021, 8, 639184. [CrossRef] Nanomaterials 2021, 11, 2775 16 of 16 11. Bonoiu, A.C.; Mahajan, S.D.; Ding, H.; Roy, I.; Yong, K.-T.; Kumar, R.; Hu, R.; Bergey, E.J.; Schwartz, S.A.; Prasad, P.N. Nanotechnology approach for drug addiction therapy: Gene silencing using delivery of gold nanorodsiRNA nanoplex in dopaminergic neurons. Proc. Natl. Acad. Sci. USA 2009, 106, 5546–5550. [CrossRef] 12. Bai, X.; Wang, Y.; Song, Z.; Feng, Y.; Chen, Y.; Zhang, D.; Feng, L. The Basic Properties of Gold Nanoparticles and their Applications in Tumor Diagnosis and Treatment. Int. J. Mol. Sci. 2020, 21, 2480. [CrossRef] 13. Li, W.; Cao, Z.H.; Liu, R.; Liu, L.; Lia, H.; Li, X.; Chen, Y.; Lu, C.H.; Liu, Y. AuNPs as an important inorganic nanoparticle applied in drug carrier systems. Artif. Cells Nanomed. 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A Lipid-Coated Nanoconstruct Composed of Gold Nanoparticles Noncovalently Coated with Small Interfering RNA: Preparation, Purification and Characterization

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nanomaterials Article A Lipid-Coated Nanoconstruct Composed of Gold Nanoparticles Noncovalently Coated with Small Interfering RNA: Preparation, Purification and Characterization † † † Anna V. Epanchintseva , Julia E. Poletaeva , Ilya S. Dovydenko , Boris P. Chelobanov, Dmitrii V. Pyshnyi , Elena I. Ryabchikova * and Inna A. Pyshnaya * Institute of Chemical Biology and Fundamental Medicine SB RAS, 630090 Novosibirsk, Russia; annaepanch@niboch.nsc.ru (A.V.E.); fabaceae@yandex.ru (J.E.P.); dovydenko.il@gmail.com (I.S.D.); boris.p.chelobanov@gmail.com (B.P.C.); pyshnyi@niboch.nsc.ru (D.V.P.) * Correspondence: lenryab@yandex.ru (E.I.R.); pyshnaya@niboch.nsc.ru (I.A.P.); Tel.: +7-(383)-363-5163 (E.I.R. & I.A.P.) † These authors contributed equally to this work. Abstract: 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 Citation: Epanchintseva, A.V.; for their multistep fabrication were found. All steps of this process and MLNC purification were Poletaeva, J.E.; Dovydenko, I.S.; verified using dynamic light scattering, transmission electron microscopy, and UV-Vis spectroscopy. Chelobanov, B.P.; Pyshnyi, D.V.; Ryabchikova, E.I.; Pyshnaya, I.A. A Factors influencing the efficiency of nanocomposite assembly, colloidal stability, and purification Lipid-Coated Nanoconstruct quality were identified. These data made it possible to optimize the fabrication of target MLNCs Composed of Gold Nanoparticles bearing small interfering RNA and to substantially improve end product quality via an increase in Noncovalently Coated with Small its homogeneity and a decrease in the amount of incomplete nanoconstructs. We believe that the Interfering RNA: Preparation, proposed approaches and methods will be useful for researchers working with lipid nanoconstructs. Purification and Characterization. Nanomaterials 2021, 11, 2775. https:// Keywords: gold nanoparticles; siRNA; noncovalent adsorption; lipid enveloping; multilayer nanocon- doi.org/10.3390/nano11112775 structs for siRNA preparation and purification Academic Editor: Paulo Cesar De Morais 1. Introduction Received: 1 October 2021 Accepted: 18 October 2021 Nucleic-acid therapeutics have tremendous therapeutic potential for the treatment of Published: 20 October 2021 many diseases. Nonetheless, the number of such therapeutics approved for clinical use is still low [1]. First of all, the reason is the nature of nucleic acids, which are macromolecules Publisher’s Note: MDPI stays neutral with a high negative charge and therefore are unable to penetrate into the cell on their with regard to jurisdictional claims in own [2]. In addition, nucleic acids are very sensitive to nucleases, as evidenced by the published maps and institutional affil- low stability of nucleic acids in physiological fluids [3]. These obstacles can be overcome iations. by (i) the development of modifications of nucleic acids and (ii) the creation of delivery systems that ensure not only penetration into the cell but also longer half-life of the nucleic acid formulation in the human body. During the past several decades, a variety of systems of nucleic acid delivery have Copyright: © 2021 by the authors. been proposed [4–8]. A distinct field of research and development of these systems is the Licensee MDPI, Basel, Switzerland. use of solid (metal) nanoparticles enclosed in a shell and serving as a carrier of a nucleic This article is an open access article acid. For instance, there are reports on the efficient delivery of small interfering RNA distributed under the terms and (siRNA) via gold nanoparticles (AuNPs) [9] and selenium nanoparticles [10] coated with conditions of the Creative Commons chitosan and gold nanorods covered with two layers of polyelectrolytes [11]. Attribution (CC BY) license (https:// Our study was aimed at creating multilayer nanoconstructs (MLNCs) based on spher- creativecommons.org/licenses/by/ ical AuNPs on whose surface an siRNA layer is adsorbed and surrounded by a lipid 4.0/). Nanomaterials 2021, 11, 2775. https://doi.org/10.3390/nano11112775 https://www.mdpi.com/journal/nanomaterials Nanomaterials 2021, 11, x 2 of 16 Nanomaterials 2021, 11, 2775 2 of 16 (siRNA) via gold nanoparticles (AuNPs) [9] and selenium nanoparticles [10] coated with chitosan and gold nanorods covered with two layers of polyelectrolytes [11]. Our study was aimed at creating multilayer nanoconstructs (MLNCs) based on spherical AuNPs on whose surface an siRNA layer is adsorbed and surrounded by a lipid envelope (Figure 1). The choice of AuNPs as the basis for this nanocomposite is due to envelope (Figure 1). The choice of AuNPs as the basis for this nanocomposite is due to their unique physicochemical properties and good biocompatibility [12–14]. their unique physicochemical properties and good biocompatibility [12–14]. Figure 1. The fabrication and structure of the desired MLNC. Figure 1. The fabrication and structure of the desired MLNC. In the present study, the fabrication of an MLNC includes several steps: preparation In the present study, the fabrication of an MLNC includes several steps: preparation of of core nanoparticles (AuNP-siRNA) and a lipid film, assembly of the MLNC, and purifi- core nanoparticles (AuNP-siRNA) and a lipid film, assembly of the MLNC, and purification cation and concentration of the nanoconstruct particles. One of the previously developed and concentration of the nanoconstruct particles. One of the previously developed versions versions of an MLNC—whose lipid envelope is doped with peptide (LR)4G conjugated of an MLNC—Whose lipid envelope is doped with peptide (LR) G conjugated with stearic with stearic acid—successfully delivers siRNA into cells and ensures a release of the acid—Successfully delivers siRNA into cells and ensures a release of the siRNA cargo, as siRNA cargo, as confirmed by the suppression of reporter protein expression (green fluo- confirmed by the suppression of reporter protein expression (green fluorescent protein; rescent protein; GFP) [15]. In that study, we showed the feasibility of siRNA delivery via GFP) [15]. In that study, we showed the feasibility of siRNA delivery via constructs constructs containing lipid-coated AuNPs [15]. On the other hand, designing formulations containing lipid-coated AuNPs [15]. On the other hand, designing formulations intended intended for clinical use requires higher nanoconstruct quality, which can be achieved by for clinical use requires higher nanoconstruct quality, which can be achieved by improving improving the method of MLNC fabrication and will enhance the efficiency of siRNA de- the method of MLNC fabrication and will enhance the efficiency of siRNA delivery into livery into human cells. human cells. The second aim of this work was to optimize the conditions of MLNC synthesis that The second aim of this work was to optimize the conditions of MLNC synthesis yield the most homogeneous suspensions free of empty lipid particles, naked core nano- that yield the most homogeneous suspensions free of empty lipid particles, naked core particles, and aggregates of core nanoparticles. To increase the efficiency of MLNC syn- nanoparticles, and aggregates of core nanoparticles. To increase the efficiency of MLNC thesis, we varied the conditions of all steps of this process on the basis of our previously synthesis, we varied the conditions of all steps of this process on the basis of our previously obt obtained ained and andpublish published ed da data ta abo about ut the st theabil stability ity and properti and properties es of nanoconstructs. In pa of nanoconstructs. In r- ticular, we varied the ratio of core nanoparticles to a lipid mixture; an optimal buffer was particular, we varied the ratio of core nanoparticles to a lipid mixture; an optimal buffer chosen t was chosen hat e that nable enables s effect efi fective ve wrapping wrapping of t of hethe core cor n eananoparticles noparticles wit with h a alip lipid id eenvelope; nvelope; and op and optimal timal c conditions onditions we wer re fo e found und for t for the he fr fractionation actionation and and p purification urification o of f t the he MLNC MLNCs. s. 2. Materials and Methods 2. Materials and Methods 2.1. Chemicals 2.1. Chemicals Tetrachloroauric acid trihydrate (HAuCl 3H O) was purchased from Aurat (Moscow, 4 2 Tetrachloroauric acid trihydrate (HAuCl4·3H2O) was purchased from Aurat (Mos- Russia), and RNA phosphoramidites for oligoribonucleotide synthesis were acquired from cow, Russia), and RNA phosphoramidites for oligoribonucleotide synthesis were ac- Sigma-Aldrich (Hamburg, Germany). Sodium chloride (NaCl) and magnesium sulfate quired from Sigma-Aldrich (Hamburg, Germany). Sodium chloride (NaCl) and magne- (MgSO ) were bought from Honeywell (Seelze, Germany) and sodium citrate dihydrate sium sulfate (MgSO4) were bought from Honeywell (Seelze, Germany) and sodium citrate (Na C H O 2H O) from Fluka (Buchs, Switzerland). Magnesium acetate tetrahydrate 3 6 5 7 2 dihydrate (Na3C6H5O7·2H2O) from Fluka (Buchs, Switzerland). Magnesium acetate tetra- [Mg(CH COO) 4H O] was acquired from VWR International LLC (Radnor, PA, USA), 3 2 2 hydrate [Mg(CH3COO)2·4H2O] was acquired from VWR International LLC (Radnor, PA, whereas egg phosphatidylcholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine USA), whereas egg phosphatidylcholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanola- (DOPE) was obtained from Avanti (Alabaster, AL, USA). 2-[[4-Dodecylamino-6-oleylamino- mine (DOPE) was obtained from Avanti (Alabaster, AL, USA). 2-[[4-Dodecylamino-6- 1,3,5-triazine-2yl]-(2-hydroxyethyl)amino]ethanol (DOME2) was synthetized as described oleylamino-1,3,5-triazine-2yl]-(2-hydroxyethyl)amino]ethanol (DOME2) was synthetized in ref. [13]. Disodium phosphate dihydrate (NaH PO 2H O) and monosodium phos- 2 4 2 as described in ref. [13]. Disodium phosphate dihydrate (NaH2PO4·2H2O) and monoso- phate dodecahydrate (Na HPO 12H O) were purchased from Reatex (Moscow, Russia) 2 4 2 dium phosphate dodecahydrate (Na2HPO4·12H2O) were purchased from Reatex (Mos- and uranyl acetate from SPI (West Chester, PA, USA). Peptide NH -(RL) G-C(O)NH 2 4 2 cow, Russia) and uranyl acetate from SPI (West Chester, PA, USA). Peptide NH2-(RL)4G- 5CF COOH was acquired from Diapharm (Lyubertsy, Russia). Sodium acetate trihydrate (CH COONa3H O), acetic acid (CH COOH), and sucrose (C H O ) were bought from 3 2 3 12 22 11 Panreac (Barcelona, Spain), whereas cesium chloride (CsCl) and glycerol (C H O ) were ob- 3 8 3 Nanomaterials 2021, 11, 2775 3 of 16 tained from Applichem (Darmstadt, Germany), and trichloromethane (chloroform, CHCl ) and methanol (CH OH) were purchased from Reachem (Moscow, Russia). AlamarBlue™ Cell Viability Reagent was acquired from Invitrogen (Waltham, MA, USA). Water was purified by means of a Simplicity 185 water purification system (Millipore, Burlington, MA, USA) and had a resistivity of 18.2 MWcm at 25 C. 2.2. Preparation of Core Nanoparticles To obtain core nanoparticles (AuNP-siRNA), siRNA was used that suppresses GFP synthesis in cultured cells stably expressing this protein. The sequence of the siRNA was 0 0 as follows: sense strand, 5 -CAAGCUGACCCUGAAGUUCTT; and antisense strand, 5 - GAACUUCAGGGUCAGCUUGTT [16]. The synthesis of this siRNA is described in detail in our previous work [17]. The AuNPs were synthesized using a previously published technique [18]. The core nanoparticles, which are AuNPs noncovalently covered with siRNA, were fabricated as described elsewhere [15,19], with some modifications: 4 mM citrate–stabilized AuNPs (size 12 1 nm according to transmission electron microscopy [TEM] or 17.3 2.1 nm according to dynamic light scattering [DLS] analysis; a zeta potential of 33.6  2.0 mV according to DLS) at a concentration of 3.6  10 M were incubated with 0.72 M siRNA for 22 h at room temperature in the presence of either 5 or 10 mM NaCl and either Mg(CH COO) or MgSO . The resultant suspensions of core nanoparticles were cen- 3 2 4 trifuged, the supernatant was discarded, and the pellet containing the core nanoparticles was washed with 1 mL of 4 mM sodium citrate dihydrate. The size and monodispersity of the obtained core nanoparticles were determined using TEM (13  1 nm) and DLS (25 9 nm). The zeta potential of the core nanoparticles proved to be44 1 mV, and the polydispersity index (PDI) of the suspension was 0.212 0.011. The core nanoparticles had a surface plasmon resonance maximum at 520 nm, and the density of siRNA molecules on the AuNP surface was 29  6. 2.3. Preparation of a Lipid Film The lipid film was prepared as described before [15], with modifications: 90 L of 1 mM egg phosphatidylcholine and DOPE in a CHCl /CH OH mixture (1:1) and 10 L 3 3 of 1 mM DOME2 in CHCl were added to 1 mL of CHCl in a 10 mL round-bottom flask. 3 3 Next, the solvent was evaporated either at 25 C and 6 mmHg or without thermostatting at 12 mmHg. The resultant lipid film was then dried in vacuum in a desiccator to remove traces of the organic solvents, after which the flask with the film was incubated at 18 C for 16 h. All the procedures for obtaining the lipid film were carried out in an argon atmosphere. 2.4. Synthesis of a Peptide Conjugate with Stearic Acid A conjugate of a peptide [(RL) G-NH ] with stearic acid [Str-(RL) G-NH ] for doping 4 2 4 2 the lipid envelope was synthesized as described previously [15]. 2.5. Assembly of the MLNC The procedure was performed as described earlier [15], with slightly modified reaction conditions, as described below. The MLNCs were obtained in two stages: First, a buffer with pH 4.5 was applied to the surface of the lipid film: either (1) 0.9 mL of H O and 31 L of NaH PO (1.00 M, 0.10 M, or 0.01 M) or (2) 0.825 mL of H O; to the 2 2 4 2 buffer, we added 100 L of a suspension of the core nanoparticles (2.5 pmol in terms of gold) in 6 mM CH COOH. The reaction mixture of the core nanoparticles and lipid film was sonicated at 90 W for 15 min at 25 C. The expected result of this step is a suspension of the core nanoparticles bearing a layer of noncovalently attached siRNA enclosed in a lipid envelope. Second stage: the pH of the suspension containing the core nanoparticles carrying the lipid envelope was adjusted to 7.4 by the addition of either (1) 69 L of 1.00, 0.10, or 0.01 M Nanomaterials 2021, 11, 2775 4 of 16 Na HPO or (2) 100 L of 3 mM CH COONa. Then, 10 L of 1 mM stearic-acid-conjugated 2 4 3 peptide, Str-(LR) G, was introduced. The reaction mixture was sonicated (90 W) for 5 min at 25 C. The size and monodispersity of the obtained MLNCs were verified using TEM and DLS. The MLNCs had a surface plasmon resonance maximum at 534 nm. Thus, we obtained a suspension of MLNCs whose envelope was doped with a peptide. For brevity, hereafter, these nanoparticles are referred to as “MLNCs,” without a mention of the peptide. 2.6. Purification of the MLNCs by Banding Centrifugation Fractionation of the MLNCs was carried out either {1} in a cesium chloride gradient or {2} in solutions of glycerol or sucrose. {1} In a 1.5 mL test tube, we sequentially layered 500 L each of an aqueous 61.7% CsCl solution ( = 1.789 g/cm ,  = 1.238 9 cP) and an aqueous 10.8% CsCl solution ( = 1.0804 g/cm ,  = 0.966 cP) and then 50 L of the MLNC suspension; its colloidal stability was examined by means of changes in the color of the MLNC layer. {2} In a 15 mL test tube, we layered 500–1000 L of the MLNC suspension on top of 10 mL of (i) an aqueous 75% glycerol solution ( = 41 cP) or (ii) an aqueous 26% sucrose solution in 1 mM phosphate buffer (pH was altered) ( = 1.1082 g/mL,  = 2.223 cP) or (iii) an aqueous 58% sucrose solution in 1 mM phosphate buffer ( = 1.267 g/mL, = 42.8 cP), followed by centrifugation at 25 C in the range of 1000–8000 g for 15–90 min. Fractions of different colors were collected into separate 1.5 mL tubes and were subjected to purification to remove excess glycerol or sucrose via dialysis or centrifugation. After that, all the fractions were analyzed using DLS and TEM. After the fractionation in sucrose, the samples with or without an added equal volume of 1 mM phosphate buffer were centrifuged in 1.5 mL tubes at 2000 g for 15, 30, or 60 min, the supernatant was discarded, and the resulting MLNCs were analyzed via DLS, TEM, and UV-Vis spectroscopy. After the fractionation in glycerol, dialysis was performed either in Centricon Plus-70 3 kDa, Merck KGaA (Darmstadt, Germany) or in screw cap 1.5 mL tubes at 25 C for 16–72 h against 1, 3, or 10 mM phosphate buffer using either (1) membranes for extruders with a pore diameter of 30 or 100 nm, Whatman (Maidstone, UK) or (2) single-layer 3.5 or 10 kDa dialysis bags, Thermo Fisher Scientific (Göteborg, Sweden). 2.7. Examination of the Composition and Quality of the Samples 2.7.1. Optical Extinction Spectra To verify colloidal stability and to determine gold concentration, we performed UV-Vis spectroscopy. Optical adsorption spectra of the core nanoparticles and all preparations of MLNCs (Figure 2) were recorded on a Clariostar plate fluorimeter, BMG Labtech (Orten- berg, Germany) in the range 400–800 nm according to the manufacturer ’s instructions. The absorption maximum of all versions of MLNC preparations was found to be shifted by 12 nm relative to AuNPs and core nanoparticles; this shift emerged after the lipid envelope formation and was not affected by other experimental conditions. Preservation of colloidal stability of the samples during the procedure was confirmed by the absence of a peak in the range 600–700 nm. Nanomaterials 2021, 11, 2775 5 of 16 Nanomaterials 2021, 11, x 5 of 16 Figure 2. “Typical” spectra of the obtained samples of AuNPs, core nanoparticles, and MLNCs. The Figure 2. “Typical” spectra of the obtained samples of AuNPs, core nanoparticles, and MLNCs. vertical lines indicate absorption maxima for AuNPs and core nanoparticles (520 nm, left-hand line) The vertical lines indicate absorption maxima for AuNPs and core nanoparticles (520 nm, left-hand and for MLNCs (532 nm, right-hand line). For clarity, we present the spectra of the samples at con- line) and for MLNCs (532 nm, right-hand line). For clarity, we present the spectra of the samples at centrations that ensure resolution of the lines in the graph. concentrations that ensure resolution of the lines in the graph. The yield of MLNCs was calculated from optical density of the final suspension at The yield of MLNCs was calculated from optical density of the final suspension 520 nm using the molar extinction coefficient characteristic of the original AuNPs (ε = 8.78 at 520 nm using the molar extinction coefficient characteristic of the original AuNPs × 10 L·mol/cm) [20]. The amount (mol) of AuNPs used to fabricate the core nanoparticles (" = 8.78  10 Lmol/cm) [20]. The amount (mol) of AuNPs used to fabricate the core was set to 100%. nanoparticles was set to 100%. 2.7.2. DLS 2.7.2. DLS All All samples sampleof s ocor f core e nanoparticles nanoparticles and and MLNCs MLNCwer s we ere c characterized haracterized via via D DLS LS and and TEM. TEM. DLS DLS ana analysis lysis is is employed employed f for or assessing assessing o overall verall hy hydr drod odynamic ynamic char characterist acteristics ics of of a suspen a sus- - pension of nanoparticles, whereas TEM provides information on the fine structure of sion of nanoparticles, whereas TEM provides information on the fine structure of nano- nanoparticles and its changes. These methods complement each other and, taken together, particles and its changes. These methods complement each other and, taken together, help help to exhaustively characterize samples of nanoconstructs. to exhaustively characterize samples of nanoconstructs. Suspensions of the core nanoparticles and MLNCs were analyzed using DLS by means Suspensions of the core nanoparticles and MLNCs were analyzed using DLS by of a Malvern Zetasizer Nano instrument, Malvern Instruments (Worcestershire, UK) in means of a Malvern Zetasizer Nano instrument, Malvern Instruments (Worcestershire, accordance with the manufacturer ’s instructions. This method allowed us to characterize UK) in accordance with the manufacturer’s instructions. This method allowed us to char- particle suspensions in terms of the following parameters: the hydrodynamic diameter, acterize particle suspensions in terms of the following parameters: the hydrodynamic di- PDI, and zeta () potential. The measurements were performed at least in triplicate. ameter, PDI, and zeta (ζ) potential. The measurements were performed at least in tripli- cate. 2.7.3. TEM For examination using TEM, 10 L of a suspension of the core nanoparticles or MLNCs 2.7.3. TEM was applied to a formvar film on a grid and incubated for 1 min. Then, the drop was For examination using TEM, 10 μL of a suspension of the core nanoparticles or aspirated with a pipettor, and without drying, the grid was placed on a drop of uranyl MLNCs was applied to a formvar film on a grid and incubated for 1 min. Then, the drop acetate for 10 s; the excess liquid was removed with filter paper. All TEM samples were was aspirated with a pipettor, and without drying, the grid was placed on a drop of uranyl prepared identically; at least five grid cells were examined in different parts of each grid. acetate for 10 s; the excess liquid was removed with filter paper. All TEM samples were The suspensions of the core nanoparticles and MLNCs were investigated under a JEM prepared identically; at least five grid cells were examined in different parts of each grid. 1400 transmission electron microscope, Jeol (Tokyo, Japan) equipped with a Veleta digital The suspensions of the core nanoparticles and MLNCs were investigated under a JEM camera, EM SIS (Muenster Germany). Particle sizes were determined using iTEM software, 1400 transmission electron microscope, Jeol (Tokyo, Japan) equipped with a Veleta digital version 5.2, EM SIS (Muenster, Germany). camera, EM SIS (Muenster Germany). Particle sizes were determined using iTEM soft- ware, version 5.2, EM SIS (Muenster, Germany). Nanomaterials 2021, 11, 2775 6 of 16 2.8. Cytotoxicity Assays of Glycerol and Sucrose To this end, SC-1 R780 fibroblasts were cultured in the IMDM medium, Gibco (Grand Island, NY, USA) supplemented with 10% of fetal calf serum (Gibco), penicillin (100 U/mL), and streptomycin (100 g/mL) in a humidified atmosphere containing 5% of CO at 37 C. The cytotoxicity assay was performed using the AlamarBlue test [21]. 2.9. Statistical Analysis Each experiment was conducted independently at least three times. Data are presented as mean  standard deviation from at least three independent experiments. 3. Results 3.1. The Mechanism of MLNC Formation The formation of a lipid envelope around the core nanoparticle is mediated by elec- trostatic interaction between protonated amino groups of the lipid (DOME2, which is a component of the lipid film) and negatively charged phosphate groups of the siRNA on the surface of the core nanoparticle. Because DOME2 is protonated at a pH below 7, the assembly of the nanoconstructs was carried out in an acidic medium. This strategy necessitated selecting a buffer that makes it easy to switch DOME2 to either a charged (protonated) state or a neutral (deprotonated) state and ensures colloidal stability of the core nanoparticles. We believe that choosing such a buffer will reduce the aggregation of MLNC particles and aggregation of “naked” core nanoparticles. To form a lipid envelope around the core nanoparticle, a suspension of the core nanoparticles in an acidic buffer was applied to the prepared lipid film, and the reaction mixture was sonicated (Figure 3A). The lipid film, which carries the core nanoparticles adsorbed on the surface, gets fragmented, and the core nanoparticles end up in a lipid envelope. The sizes of the forming particles are different, as are the numbers of core nanoparticles enclosed in a single shell. Consequently, the resulting suspension contains structurally diverse desired MLNCs (“target MLNCs”: Figure 3B, panel 1), aggregates of the core nanoparticles surrounded by a thin lipid envelope (Figure 3B, panel 2), lipid particles and empty vesicles (Figure 3B, panel 3), lipid film fragments of various sizes with adhered core nanoparticles (Figure 3B, panel 4), and naked core nanoparticles, both stand-alone and aggregated (Figure 3B, panel 5). It is obvious that the ideal end product should consist mainly of stand-alone MLNCs each containing one core nanoparticle. The presented mechanism of MLNC formation makes this outcome a pipe dream, mainly owing to the heterogeneity of adsorption of the core nanoparticles on the lipid film. Therefore, our efforts were focused on obtain- ing suspensions with the highest proportion of stand-alone MLNCs containing 1–10 core nanoparticles and a distinct electron-transparent envelope; such nanoconstructs are here- inafter referred to as “target MLNCs” (Figure 3B, panel 1). Meanwhile, we strived to reduce the concentrations of empty lipid particles, aggregates of core nanoparticles enclosed in a thin lipid envelope, and naked (unenveloped) core nanoparticles (Figure 3B, panels 2–5). It should be noted that we always assessed the effect of one or another modification of the protocol by means of changes in the characteristics of the end product: the final suspension of MLNCs. Nanomaterials 2021, 11, 2775 7 of 16 Nanomaterials 2021, 11, x 7 of 16 Figure 3. (A) The scheme of MLNC formation after the application of a suspension of the core na- Figure 3. (A) The scheme of MLNC formation after the application of a suspension of the core noparticles to the lipid film and sonication. (B) Morphology of the particles in the MLNC prepara- nanoparticles to the lipid film and sonication. (B) Morphology of the particles in the MLNC prepa- tions. Panel 1: different subtypes of a “target MLNC” containing 1 to 10 electron-dense core nano- rations. Panel 1: Different subtypes of a “target MLNC” containing 1 to 10 electron-dense core particles; Panel 2: aggregates of the core nanoparticles surrounded by a thin lipid envelope; Panel nanoparticles; Panel 2: Aggregates of the core nanoparticles surrounded by a thin lipid envelope; 3: empty lipid particles; Panel 4: fragments of the lipid film with adhered core nanoparticles; Panel Panel 5: “naked” cor 3: Empty lipid e nanoparticles. The scale particles; Panel 4: Fragments bars correspond t of the lipid o 50 nm. Nega film with adher tive edstaining with uranyl core nanoparticles; acetate followed by TEM. Panel 5: “Naked” core nanoparticles. The scale bars correspond to 50 nm. Negative staining with uranyl acetate followed by TEM. 3.2. Assembly of the Core Nanoparticles: AuNP-siRNA 3.2. Assembly of the Core Nanoparticles: AuNP-siRNA The first step in the fabrication of MLNCs is the synthesis of core nanoparticles The first step in the fabrication of MLNCs is the synthesis of core nanoparticles (AuNP- (AuNP-siRNA); we have described this procedure in detail previously [15,22,23]. For en- siRNA); we have described this procedure in detail previously [15,22,23]. For enveloping veloping the core nanoparticles with the siRNA, a suitable buffer was experimentally se- the core nanoparticles with the siRNA, a suitable buffer was experimentally selected, lected, phosphate buffer, which enabled easy switching from pH 5 to pH 7.4, which cor- phosphate buffer, which enabled easy switching from pH 5 to pH 7.4, which corresponds responds to physiological pH. It is important that this buffer ensured colloidal stability of to physiological pH. It is important that this buffer ensured colloidal stability of the core the core nanoparticles throughout the entire experiment on MLNC fabrication. nanoparticles throughout the entire experiment on MLNC fabrication. During the preparation of MLNCs, it became apparent that the lipid particles, which During the preparation of MLNCs, it became apparent that the lipid particles, which contained lipid DOME2, had negative ζ potential in acidic phosphate buffer, indicating contained lipid DOME2, had negative  potential in acidic phosphate buffer, indicating adsorption of phosphate anions on the surface of the lipid film. Accordingly, the phos- adsorption of phosphate anions on the surface of the lipid film. Accordingly, the phosphate phate component of the buffer will compete with the core nanoparticles during their ad- component of the buffer will compete with the core nanoparticles during their adsorption sorption onto the lipid film, thereby inevitably decreasing the efficiency of the enveloping onto the lipid film, thereby inevitably decreasing the efficiency of the enveloping process. process. At first glance, the simplest solution seems to be to change the buffer, but this At first glance, the simplest solution seems to be to change the buffer, but this approach approach can diminish colloidal stability of the core nanoparticles. We reduced the phos- can diminish colloidal stability of the core nanoparticles. We reduced the phosphate anion phate anion concentration from 0.100 to 0.001 M, which increased the efficiency of MLNC concentration from 0.100 to 0.001 M, which increased the efficiency of MLNC formation formation (Figure 4) and decreased the PDI (Table 1). (Figure 4) and decreased the PDI (Table 1). Nanomaterials 2021, 11, x 8 of 16 Table 1. Physicochemical parameters of MLNCs at different concentrations of phosphate buffer (PB). MLNC Sample ID PB Concentration, mM PDI Hydrodynamic Diameter, nm 1 100 0.203 ± 0.012 749.2 ± 177 Nanomaterials 2021, 11, 2775 8 of 16 2 10 0.256 ± 0.010 169 ± 88 3 3 0.275 ± 0.015 137.7 ± 65 4 1 0.209 ± 0.009 158.7 ± 74 Figure 4. Ultrastructure of the obtained MLNCs. (A) Assembly in 100 mM phosphate buffer, (B) assembly in 10 mM phos- Figure 4. Ultrastructure of the obtained MLNCs. (A) Assembly in 100 mM phosphate buffer, (B) assembly in 10 mM phate buffer, and (C) assembly in 1 mM phosphate buffer. The scale bars correspond to 100 nm. Negative staining with a phosphate buffer, and (C) assembly in 1 mM phosphate buffer. The scale bars correspond to 100 nm. Negative staining with 0.5% uranyl acetate solution followed by TEM. a 0.5% uranyl acetate solution followed by TEM. By lowering the buffer concentration, we reduced the amount of phosphate anions Table 1. Physicochemical parameters of MLNCs at different concentrations of phosphate buffer (PB). adsorbed on the lipid film surface; however, it was necessary to neutralize the effect of the remaining ones. For this purpose, it was decided to add a divalent cation that would act MLNC Sample ID PB Concentration, mM PDI Hydrodynamic Diameter, nm as a linker between the phosphate anions adsorbed on the lipid film surface and the siRNA 1 2+100 0.203 0.012 749.2  177 on the surface of AuNPs. Mg was chosen and did not lead to “adhesion” and precipita- 2 10 0.256 0.010 169  88 tion of the components of the lipid film and core nanoparticles, nor does it catalyze phos- 3 3 0.275 0.015 137.7  65 phodiester bond hydrolysis in siRNA. Nevertheless, which salt can be used in this con- 4 1 0.209 0.009 158.7  74 text? It is known that the type of anion has a pronounced effect on colloidal stability of core nanoparticles [24–26]. Accordingly, we introduced either magnesium acetate or mag- By lowering the buffer concentration, we reduced the amount of phosphate anions nesium sulfate into the reaction mixture. adsorbed on the lipid film surface; however, it was necessary to neutralize the effect of During the synthesis of the core nanoparticles, the influence of Mg(CH3COO)2 or the remaining ones. For this purpose, it was decided to add a divalent cation that would MgSO4 at 0.1 or 0.4 mM added to the reaction mixture was determined by means of phys- act as a linker between the phosphate anions adsorbed on the lipid film surface and the icochemical characteristics (Table 2) and alterations of colloidal stability of the core nano- 2+ siRNA on the surface of AuNPs. Mg was chosen and did not lead to “adhesion” and particle suspension. Acetate ions at both concentrations affected the colloidal stability neg- precipitation of the components of the lipid film and core nanoparticles, nor does it catalyze atively. The addition of magnesium ions in the form of 0.1 mM sulfate “improved” hy- phosphodiester bond hydrolysis in siRNA. Nevertheless, which salt can be used in this drodynamic parameters of the core nanoparticles and did not alter colloidal stability for context? It is known that the type of anion has a pronounced effect on colloidal stability several days. At 0.4 mM, the impact of this salt was negative. of core nanoparticles [24–26]. Accordingly, we introduced either magnesium acetate or magnesium sulfate into the reaction mixture. Table 2. Physicochemical characteristics of the core nanoparticles in phosphate buffer containing different magnesium During the synthesis of the core nanoparticles, the influence of Mg(CH COO) or 3 2 salts. MgSO at 0.1 or 0.4 mM added to the reaction mixture was determined by means of Sample Magnesium Salt ζ Potential, mV Hydrodynamic Diameter, nm PDI physicochemical characteristics (Table 2) and alterations of colloidal stability of the core Core nanoparticles nanoparticle none suspension.−Acetate 44 ± 1 ions at both concentrations 25 ± 9 affected the colloidal 0.212 ± 0. stability 011 negatively. The addition of magnesium ions in the form of 0.1 mM sulfate “improved” Core nanoparticles 0.1 mM Mg(Ac)2 −45 ± 1 27 ± 11 0.416 ± 0.041 hydrodynamic parameters of the core nanoparticles and did not alter colloidal stability for Core nanoparticles 0.1 mM MgSO4 −39 ± 1 25 ± 9 0.266 ± 0.050 several days. At 0.4 mM, the impact of this salt was negative. Core nanoparticles 0.4 mM MgSO4 −40 ± 2 90 ± 85 0.565 ± 0.316 Table 2. Physicochemical characteristics of the core nanoparticles in phosphate buffer containing different magnesium salts. Having found the optimal concentration of MgSO4 (0.1 mM) for obtaining the core nanoparticles, we began to vary the concentration of NaCl, the presence of which is nec- Sample Magnesium Salt  Potential, mV Hydrodynamic Diameter, nm PDI essary for denser loading of siRNA molecules onto AuNPs. AuNPs are very sensitive to Core nanoparticles none 44  1 25  9 0.212  0.011 Core nanoparticles 0.1 mM Mg(Ac) 45  1 27  11 0.416  0.041 Core nanoparticles 0.1 mM MgSO 39  1 25  9 0.266  0.050 Core nanoparticles 0.4 mM MgSO 40  2 90  85 0.565  0.316 Having found the optimal concentration of MgSO (0.1 mM) for obtaining the core nanoparticles, we began to vary the concentration of NaCl, the presence of which is nec- Nanomaterials 2021, 11, 2775 9 of 16 Nanomaterials 2021, 11, x 9 of 16 essary for denser loading of siRNA molecules onto AuNPs. AuNPs are very sensitive to monovalent anions [25], and even a small shift in their concentration can affect colloidal monovalent anions [25], and even a small shift in their concentration can affect colloidal stability of the resulting core nanoparticles. Previously [15], we prepared core nanoparti- stability of the resulting core nanoparticles. Previously [15], we prepared core nanoparti- cles at 10 mM NaCl (parameters of the obtained preparations of MLNCs: hydrodynamic cles at 10 mM NaCl (parameters of the obtained preparations of MLNCs: hydrodynamic diameter of 205  100 nm and PDI of 0.186), but in the present work, we reduced the diameter of 205 ± 100 nm and PDI of 0.186), but in the present work, we reduced the NaCl NaCl concentration in half, which increased suspension homogeneity and reduced the concentration in half, which increased suspension homogeneity and reduced the size of size of MLNCs (hydrodynamic diameter became 128  54 nm, and PDI became 0.151). MLNCs (hydrodynamic diameter became 128 ± 54 nm, and PDI became 0.151). The change The change in NaCl concentration did not lead to a noticeable alteration of the end prod- in NaCl concentration did not lead to a noticeable alteration of the end products’ compo- ucts’ composition according to TEM; they contained MLNCs with different numbers of sition according to TEM; they contained MLNCs with different numbers of core nanopar- core nanoparticles as well as lipid particles, empty lipid vesicles, aggregates of the core ticles as well as lipid particles, empty lipid vesicles, aggregates of the core nanoparticles, nanoparticles, and a small percentage of naked core nanoparticles (Figure 5). In subsequent and a small percentage of naked core nanoparticles (Figure 5). In subsequent experiments, experiments, the reaction mixture version containing 0.1 mM MgSO and 5 mM NaCl was the reaction mixture version containing 0.1 mM MgSO4 and 5 mM NaCl was utilized to utilized to synthesize the core nanoparticles. synthesize the core nanoparticles. Figure 5. Ultrastructure of the MLNCs obtained in the presence of 0.1 mM MgSO4. (A) MLNCs Figure 5. Ultrastructure of the MLNCs obtained in the presence of 0.1 mM MgSO . (A) MLNCs prepared via the addition of 10 mM NaCl or (B) 5 mM NaCl. The scale bars correspond to 200 nm. prepared via the addition of 10 mM NaCl or (B) 5 mM NaCl. The scale bars correspond to 200 nm. Negative staining with a 0.5% uranyl acetate solution followed by TEM. Negative staining with a 0.5% uranyl acetate solution followed by TEM. 3.3. Fabrication of the Lipid Film 3.3. Fabrication of the Lipid Film The lipid film was generated via evaporation of a solution of mixed lipids in a chlo- The lipid film was generated via evaporation of a solution of mixed lipids in a chloro- roform/methanol mixture under reduced pressure (6 mmHg). We found that the evapo- form/methanol mixture under reduced pressure (6 mmHg). We found that the evaporation ration rate, which can be adjusted by changing pressure, affects the quality of the resulting rate, which can be adjusted by changing pressure, affects the quality of the resulting film: an film: an increase in pressure from 6 to 12 mmHg and elimination of flask thermostatting increase in pressure from 6 to 12 mmHg and elimination of flask thermostatting improved improved the homogeneity of the lipid film and facilitated its interaction with the core the homogeneity of the lipid film and facilitated its interaction with the core nanoparticles. nanoparticles. Accordingly, more homogeneous end products with a higher concentration Accordingly, more homogeneous end products with a higher concentration of MLNCs were of MLNCs were obtained. For instance, the hydrodynamic diameter of MLNCs decreased obtained. For instance, the hydrodynamic diameter of MLNCs decreased from 188  96 to from 144.3 18 8 ± 9 79 nm, 6 to 1 while 44.3 ± the 79 nm PDI, was whil0.259 e the  PDI 0.005 was 0. nm2and 59 ± 0. 0.250 005 and  0.006 0.25 nm, 0 ± 0. respectively 006, respec- . TEM tively. TE analysis M analy of these sis of the sample se samp s revealed les reno veale app d no ap reciable preciab differ le differences in the ences in their composition: ir compo- they contained MLNCs of various structures, empty lipid particles, aggregates of the core sition: they contained MLNCs of various structures, empty lipid particles, aggregates of nanoparticles the core nano within particle as w thin ith lipid in a t envelope, hin lipid envelope, and naked an cor d nak e nanoparticles ed core nanopart (Figur ic ele 6s ). (Figure 6). Nanomaterials 2021, 11, 2775 10 of 16 Nanomaterials 2021, 11, x 10 of 16 Figure 6. Ultrastructure of the MLNCs prepared from the lipid film synthesized under a pressure Figure 6. Ultrastructure of the MLNCs prepared from the lipid film synthesized under a pressure of of 12 mmHg (A) or 6 mmHg (B). The scale bars correspond to 200 nm. Negative staining with a 0.5% 12 mmHg (A) or 6 mmHg (B). The scale bars correspond to 200 nm. Negative staining with a 0.5% uranyl acetate solution followed by TEM. uranyl acetate solution followed by TEM. 3.4. Doping of the Lipid Envelope of MLNCs with the Peptide 3.4. Doping of the Lipid Envelope of MLNCs with the Peptide The final step in the fabrication of the target MLNCs is doping of the lipid envelope The final step in the fabrication of the target MLNCs is doping of the lipid envelope with peptide Str-(LR)4G at pH 7.4 to make sure that the nanoparticles can penetrate into with peptide Str-(LR) G at pH 7.4 to make sure that the nanoparticles can penetrate into the cell and to overcome endosomal arrest as well as to enable the release of the siRNA the cell and to overcome endosomal arrest as well as to enable the release of the siRNA into into the cytosol from the surface of the core nanoparticle. The effectiveness of this proce- the cytosol from the surface of the core nanoparticle. The effectiveness of this procedure dure has been demonstrated by us previously [15]. In the present work, a sample of lipid- has been demonstrated by us previously [15]. In the present work, a sample of lipid-coated coated core nanoparticles obtained at pH 4.5 via sonication was transferred to a medium core nanoparticles obtained at pH 4.5 via sonication was transferred to a medium with pH with pH 7.5, after which the peptide was added, and the mixture was sonicated for 5 min 7.5, after which the peptide was added, and the mixture was sonicated for 5 min at 25 C. at 25 °C. It turned out that the doping of the lipid envelope with Str-(LR) G affects physic- It turned out that the doping of the lipid envelope with Str-(LR)4G affects physico- ochemical parameters of the resultant MLNCs: the addition of the peptide yielded an chemical parameters of the resultant MLNCs: the addition of the peptide yielded an in- increase in the hydrodynamic diameter from 118.4  44.27 to 235.1  100.3 nm, while the crease in the hydrodynamic diameter from 118.4 ± 44.27 to 235.1 ± 100.3 nm, while the PDI PDI was 0.1942 and 0.1854 for the MLNCs without doping and after the doping, respec- was 0.1942 and 0.1854 for the MLNCs without doping and after the doping, respectively. tively. Structural analysis of the end product using TEM did not uncover any noticeable Structural analysis of the end product using TEM did not uncover any noticeable changes changes in its composition and in particle structure of the MLNCs after the doping with in its composition and in particle structure of the MLNCs after the doping with peptide peptide Str-(LR) G. Str-(LR)4G. These findings allowed us to find optimal modifications of the initial protocol [15] that affor These f d theindin bestgs quality allowed of MLNCs: us to find the opt pr imal mod esence ofif0.1 icatmM ions of t MgSO he in and itial prot 5 mMoco NaCl l [15] during that affo therd t assembly he best qu of the alitcor y of MLNC e nanoparticles, s: the prlipid esence o filmf 0. synthesis 1 mM M at gSO 12 4mmHg and 5 m without M NaCl thermostatting, and the assembly of MLNCs in 1 mM phosphate buffer. The end product during the assembly of the core nanoparticles, lipid film synthesis at 12 mmHg without obtained thermostatting, under these and the conditions assembly o is herf einafter MLNCs referr in 1 mM ed to phosphate b as “optimized uffer. The en MLNCs” (Figur d product e 7). The preparations of optimized MLNCs are characterized by a hydrodynamic diameter of obtained under these conditions is hereinafter referred to as “optimized MLNCs” (Figure 152  75 nm and a PDI of 0.201  0.012. 7). The preparations of optimized MLNCs are characterized by a hydrodynamic diameter The presented modifications of the protocol of MLNC fabrication increased the ef- of 152 ± 75 nm and a PDI of 0.201 ± 0.012. ficiency of synthesis of core nanoparticles carrying the lipid envelope, but the obtained The presented modifications of the protocol of MLNC fabrication increased the effi- suspensions still contained a noticeable amount of lipid particles and vesicles, aggregates ciency of synthesis of core nanoparticles carrying the lipid envelope, but the obtained sus- of the core nanoparticles within a thin lipid envelope, and naked core nanoparticles, thus pensions still contained a noticeable amount of lipid particles and vesicles, aggregates of necessitating purification of the MLNC preparations. Given that the MLNCs, lipid parti- the core nanoparticles within a thin lipid envelope, and naked core nanoparticles, thus cles, and core nanoparticles have different densities and sizes, they can be separated using necessitating purification of the MLNC preparations. Given that the MLNCs, lipid parti- banding high-speed centrifugation. cles, and core nanoparticles have different densities and sizes, they can be separated using banding high-speed centrifugation. Nanomaterials 2021, 11, 2775 11 of 16 Nanomaterials 2021, 11, x 11 of 16 Figure 7. Ultrastructure of optimized MLNCs. The scale bar corresponds to 200 nm. Negative stain- Figure 7. Ultrastructure of optimized MLNCs. The scale bar corresponds to 200 nm. Negative ing with a 0.5% uranyl acetate solution followed by TEM. staining with a 0.5% uranyl acetate solution followed by TEM. 3.5. Fractionation of MLNCs via Centrifugation 3.5. Fractionation of MLNCs via Centrifugation Cesium chloride density gradient fractionation is the best-studied and widely used Cesium chloride density gradient fractionation is the best-studied and widely used technique for the separation of macromolecules. We fractionated optimized MLNCs in a technique for the separation of macromolecules. We fractionated optimized MLNCs in a stepwise CsCl gradient; as a consequence, the color of the sample turned from dark red to stepwise CsCl gradient; as a consequence, the color of the sample turned from dark red blue, indicating complete loss of colloidal stability of these nanoparticles in the cesium to blue, indicating complete loss of colloidal stability of these nanoparticles in the cesium chloride solution. chloride solution. After giving up on cesium chloride, for the fractionation of MLNCs, we chose an After giving up on cesium chloride, for the fractionation of MLNCs, we chose an aqueous solution of sucrose as a viscous and less “aggressive” medium. The fractionation aqueous solution of sucrose as a viscous and less “aggressive” medium. The fractionation was conducted in a homogeneous solution without a concentration gradient. The sucrose was conducted in a homogeneous solution without a concentration gradient. The sucrose concentration was selected according to calculations suggesting that target MLNCs concentration was selected according to calculations suggesting that target MLNCs should should penetrate into the viscous medium to 2 cm depth. The computation was performed penetrate into the viscous medium to 2 cm depth. The computation was performed using using a calculator [27]. To this end, the density of the nanoparticles was assumed to equal a calculator [27]. To this end, the density of the nanoparticles was assumed to equal that of gold, 19 g/cm , and we hypothesized that during the centrifugation, the less dense that of gold, 19 g/cm , and we hypothesized that during the centrifugation, the less envelope and the heavy dense core would move at different acceleration rates. In accord- dense envelope and the heavy dense core would move at different acceleration rates. In ance with the calculation results, a 58% sucrose solution was selected (ρ = 1.267 g/mL, μ = accordance with the calculation results, a 58% sucrose solution was selected ( = 1.267 g/mL, 42.8 cP). = 42.8 cP). On top of the sucrose solution, 500–1000 μL of the optimized MLNC suspension was On top of the sucrose solution, 500–1000 L of the optimized MLNC suspension layered, followed by centrifugation at 25 °C and 2000× g for 1 h. The resultant fractions was layered, followed by centrifugation at 25 C and 2000 g for 1 h. The resultant fractions were carefully collected w were carefully collected ith a dispenser. The h with a dispenser igh . The concentration high concentration of sucrosof e m sucr akeose s the makes collect the ed collected fractions unsu fractions itable for exam unsuitable for inat examination ion using TEM and acc using TEMo and rding accor ly for dingly assess for ing assessing their quality. To remove the excess sucrose, an equal volume of 1 mM phosphate their quality. To remove the excess sucrose, an equal volume of 1 mM phosphate buffer buf wa fer s a was dded added to the sa to the mp samples les of selected f of selected racti fractions ons of ML ofNCs; MLNCs; the res theur lting esulting suspens suspensions ions were wer cent e centrifuged rifuged for 1 for 5 m 15 in min at 20 at 00× 2000 g. The  g. supern The supernatant atant was d was iscarde discar d, the pellet was re ded, the pellet was sus- resuspended in 1 mM phosphate buffer, and this sample was analyzed via TEM and DLS. pended in 1 mM phosphate buffer, and this sample was analyzed via TEM and DLS. The TEM analysis of the fractions obtained via centrifugation in the 58% sucrose The TEM analysis of the fractions obtained via centrifugation in the 58% sucrose so- solution showed that the middle and upper fractions were very similar because target lution showed that the middle and upper fractions were very similar because target MLNCs constituted the bulk of each sample (Figure 8A,B). By contrast, the bottom fraction MLNCs constituted the bulk of each sample (Figure 8A,B). By contrast, the bottom fraction mainly consisted of large aggregates of the core nanoparticles enclosed in a thin lipid mainly consisted of large aggregates of the core nanoparticles enclosed in a thin lipid en- envelope and aggregates of naked core nanoparticles (Figure 8C). velope and aggregates of naked core nanoparticles (Figure 8C). Nanomaterials 2021, 11, 2775 12 of 16 Nanomaterials 2021, 11, x 12 of 16 Figure 8. Ultrastructure of the fractions of optimized MLNCs obtained via centrifugation in a 58% sucrose solution for 1 Figure 8. Ultrastructure of the fractions of optimized MLNCs obtained via centrifugation in a 58% sucrose solution for 1 h h at 25 °C and 2000× g. (A) Upper fraction, (B) middle fraction, and (C) bottom fraction. The scale bars correspond to 200 at 25 C and 2000 g. (A) Upper fraction, (B) middle fraction, and (C) bottom fraction. The scale bars correspond to 200 nm. nm. Negative staining with a 0.5% uranyl acetate solution followed by TEM. Negative staining with a 0.5% uranyl acetate solution followed by TEM. We evaluated the yield of target MLNCs after all the steps of fabrication and purifi- We evaluated the yield of target MLNCs after all the steps of fabrication and purifi- cation. For this purpose, the gold content of the target fraction was calculated from its cation. For this purpose, the gold content of the target fraction was calculated from its optical density and then divided by the amount of gold in the AuNPs used for the syn- optical density and then divided by the amount of gold in the AuNPs used for the synthesis thesis of the core nanoparticles. The yield of MLNCs (in terms of gold) in the middle frac- of the core nanoparticles. The yield of MLNCs (in terms of gold) in the middle fraction tion (Figure 8B) was 15%; the sample after the removal of excess sucrose contained 5.5% (Figure 8B) was 15%; the sample after the removal of excess sucrose contained 5.5% of of sucrose, and the hydrodynamic diameter of particles in this fraction was 195 ± 78 nm sucrose, and the hydrodynamic diameter of particles in this fraction was 195  78 nm with with a PDI of 0.173. a PDI of 0.173. The low yield of MLNCs may be related both to the low efficiency of coating of the The low yield of MLNCs may be related both to the low efficiency of coating of core nanoparticles during the fabrication of MLNCs and to degradation of the MLNC the core nanoparticles during the fabrication of MLNCs and to degradation of the MLNC sample during the fractionation because the heavy gold core can “pierce” the lipid enve- sample during the fractionation because the heavy gold core can “pierce” the lipid envelope lope during the centrifugation. Consequently, centrifugation duration was reduced to 30 during the centrifugation. Consequently, centrifugation duration was reduced to 30 min, min, which yielded four fractions represented by colored discrete rings. Characteristics of which yielded four fractions represented by colored discrete rings. Characteristics of these these fractions are given in Table 3. TEM analysis of the fractions did not reveal any ap- fractions are given in Table 3. TEM analysis of the fractions did not reveal any appreciable preciable differences in the composition and structure of their nanoparticles as compared differences in the composition and structure of their nanoparticles as compared with the with the MLNC preparations after 1 h centrifugation. MLNC preparations after 1 h centrifugation. Table 3. Physicochemical characteristics of optimized MLNCs after fractionation at 2000× g for 30 Table 3. Physicochemical characteristics of optimized MLNCs after fractionation at 2000 g min. for 30 min. Sample Hydrodynamic Diameter, nm PDI Gold Content, % Sample Hydrodynamic Diameter, nm PDI Gold Content, % Optimized MLNCs 152 ± 75 0.205 ± 0.008 - Optimized MLNCs 152  75 0.205  0.008 - Upper fraction 215 ± 108 0.198 ± 0.011 12 Upper fraction 215  108 0.198  0.011 12 Middle fraction 397 ± 163 0.160 ± 0.005 32 Middle fraction 397  163 0.160  0.005 32 Bot Bottom tom fract fraction ion 12 1259 59 ± 46  461 1 0.09 0.097 7 ± 0. 0 0.009 09 24 24 Total gold content 68 Total gold content 68 The total gold content of the four fractions was 68% of the initial gold amount in The total gold content of the four fractions was 68% of the initial gold amount in A AuNPs uNPs (u (utilized tilized f for or the sy the synthesis nthesis of core of core na nanoparticles). noparticles). One- One-thir third of d of the g the gold old (a (appar pparentl ently y in the form of naked core nanoparticles released during the disintegration of MLNCs) was in the form of naked core nanoparticles released during the disintegration of MLNCs) was di distributed stributed throughout the throughout the rest rest ((10 10 mL) mL) of of th the e sucrose so sucrose solution lution vo volume lume in in the ce the centrifuge ntrifuge tube a tube and nd was not detecta was not detectable ble by di by dir rect ob ect observation. servation. IIn n s sear earch ch of of opti optimal mal f fractionation ractionationduration, durationwe , we r reduce educe d d the the centri centri fugation fugation du duration ration to 15 min and noticed that this time enables MLNCs’ separation into two discrete fractions. to 15 min and noticed that this time enables MLNCs’ separation into two discrete frac- t The ions. The upper up fraction per fract contained ion cont13–15% ained 13 of –15% MLNCs, of MLNCs, wh which corrich corr espondsesponds to the upper to the upper fraction with the half-hour centrifugation. At the same time, an increase in the concentration of fraction with the half-hour centrifugation. At the same time, an increase in the concentra- tion of MLNCs up to 40% was observed in the middle fraction. It is this fraction (Figure 9) that we are currently testing in cell culture assays. Nanomaterials 2021, 11, 2775 13 of 16 MLNCs up to 40% was observed in the middle fraction. It is this fraction (Figure 9) that we Nanomaterials 2021, 11, x 13 of 16 are currently testing in cell culture assays. Figure 9. Ultrastructure of optimized MLNCs after 15 min fractionation in 58% sucrose and removal Figure 9. Ultrastructure of optimized MLNCs after 15 min fractionation in 58% sucrose and removal of excess sucrose. The scale bars correspond to 100 nm. Negative staining with a 0.5% uranyl acetate of excess sucrose. The scale bars correspond to 100 nm. Negative staining with a 0.5% uranyl acetate solution followed by TEM. solution followed by TEM. Our study indicates that the amount of target MLNCs in the final suspension is de- Our study indicates that the amount of target MLNCs in the final suspension is deter- termined not only by the efficiency of coating of the core nanoparticles with the lipid en- mined not only by the efficiency of coating of the core nanoparticles with the lipid envelope velope but also by MLNC preservation during the purification and concentration proce- but also by MLNC preservation during the purification and concentration procedures. We dures. We believe that the approaches that we used to improve the quality of lipid-coated believe that the approaches that we used to improve the quality of lipid-coated nanocon- nanoconstructs and the newly developed methodology will be useful to researchers cre- structs and the newly developed methodology will be useful to researchers creating similar ating similar nanoconstructs. nanoconstructs. Undoubtedly, an important factor in the proposed method is the choice of a medium Undoubtedly, an important factor in the proposed method is the choice of a medium for separating a suspension of MLNCs into fractions. In addition to sucrose and cesium for separating a suspension of MLNCs into fractions. In addition to sucrose and cesium chloride, we tested another type of viscous medium: aqueous solutions of glycerin (details chloride, we tested another type of viscous medium: aqueous solutions of glycerin (details are given in the Materials and Methods section). are given in the Materials and Methods section). The best results were obtained with the following parameters of the procedure: cen- The best results were obtained with the following parameters of the procedure: cen- trifugation in a 75% glycerol solution for 40 min at 8000× g and 25 °C with subsequent trifugation in a 75% glycerol solution for 40 min at 8000 g and 25 C with subsequent dialysis for 16 h (using membranes for extruders, pore diameter 30 nm). As in the case of dialysis for 16 h (using membranes for extruders, pore diameter 30 nm). As in the case of sucrose, in glycerol solutions, optimized MLNCs get separated into fractions (Figure 10A). sucrose, in glycerol solutions, optimized MLNCs get separated into fractions (Figure 10A). According to TEM (Figure 10B), the upper fraction contained lipid particles and vesicles According to TEM (Figure 10B), the upper fraction contained lipid particles and vesi- (hydrodynamic diameter 123 ± 55 nm, PDI = 0.366), the middle dark-red fraction contained cles (hydrodynamic diameter 123  55 nm, PDI = 0.366), the middle dark-red fraction MLNCs (hydrodynamic diameter 127 ± 40 nm, PDI = 0.279), and the bottom fraction con- contained MLNCs (hydrodynamic diameter 127  40 nm, PDI = 0.279), and the bottom sisted of large aggregates of the core nanoparticles enclosed in an “incomplete” lipid en- fraction consisted of large aggregates of the core nanoparticles enclosed in an “incomplete” velope and aggregates of core nanoparticles stuck to fragments of lipid envelopes (hydro- lipid envelope and aggregates of core nanoparticles stuck to fragments of lipid envelopes dynamic diameter 233 ± 74 nm, PDI = 0.280). (hydrodynamic diameter 233  74 nm, PDI = 0.280). We fine-tuned all stages of the fractionation of optimized MLNCs in glycerol solu- We fine-tuned all stages of the fractionation of optimized MLNCs in glycerol solutions, tions, subsequent purification via dialysis, and the concentration procedure (data not subsequent purification via dialysis, and the concentration procedure (data not shown). shown). The highest-quality end products were homogeneous, had a PDI of ~0.160, and The highest-quality end products were homogeneous, had a PDI of ~0.160, and contained contained ≥22–27% of glycerol as well as nanoparticles with a hydrodynamic diameter of 22–27% of glycerol as well as nanoparticles with a hydrodynamic diameter of 115 49 nm. 115 ± 49 nm. There were concerns that the presence of glycerol could negatively affect cell There were concerns that the presence of glycerol could negatively affect cell viability, and viability, and we performed an assay of its cytotoxicity on cultured SC-1 R780 fibroblasts. we performed an assay of its cytotoxicity on cultured SC-1 R780 fibroblasts. It turned out that when a 75% solution of glycerol is diluted 32-fold (down to 2.25% It turned out that when a 75% solution of glycerol is diluted 32-fold (down to 2.25% concentration)—which corresponds to its calculated concentration when the final MLNC concentration)—Which corresponds to its calculated concentration when the final MLNC suspension is added to the cell culture—a negative effect on cell viability is detectable. By suspension is added to the cell culture—A negative effect on cell viability is detectable. By contra contrast, st, sucrose soluti sucrose solutions ons di did d not ma not manifest nifest pronounced toxi pronounced toxicity city towa towar rd the cul d the cultur tured edfi- fibr broblasts oblasts at t at the he correspon corresponding ding di dilutions lutions (Figur (Figure 1 e 111 ). ). Nanomaterials 2021, 11, 2775 14 of 16 Nanomaterials 2021, 11, x 14 of 16 Nanomaterials 2021, 11, x 14 of 16 Figure 10. Top row: purification of optimized MLNCs via centrifugation in 75% glycerin. (A) The Figure 10. Top row: purification of optimized MLNCs via centrifugation in 75% glycerin. (A) The Figure 10. Top row: purification of optimized MLNCs via centrifugation in 75% glycerin. (A) The sample is applied to a glycerin layer; (B) separation of the sample into three fractions after centrifu- sample is applied to a glycerin layer; (B) separation of the sample into three fractions after cen- sample is applied to a glycerin layer; (B) separation of the sample into three fractions after centrifu- gation (1: upper fraction, 2: middle (target) fraction, and 3: bottom fraction). Bottom row: fractions trifugation (1: upper fraction, 2: middle (target) fraction, and 3: bottom fraction). Bottom row: gation (1: upper fraction, 2: middle (target) fraction, and 3: bottom fraction). Bottom row: fractions of optimized MLNCs obtained via centrifugation in 75% glycerol and purified via dialysis: (C) up- fractions of optimized MLNCs obtained via centrifugation in 75% glycerol and purified via dialysis: per fraction, ( of optimized MLNCs obtain D) middle fraction, and ( ed via centrifugation E) bottom fraction. The sca in 75% glycerol and p le bars correspond to urified via dialysis: ( 100 nm. C) up- (C) upper fraction, (D) middle fraction, and (E) bottom fraction. The scale bars correspond to 100 nm. per fraction, (D) middle fraction, and (E) bottom fraction. The scale bars correspond to 100 nm. Contrasting by means of a 0.5% uranyl acetate solution followed by TEM. Contrasting by means of a 0.5% uranyl acetate solution followed by TEM. Contrasting by means of a 0.5% uranyl acetate solution followed by TEM. Figure 11. Viability of SC-1 R780 fibroblasts in the presence of glycerol or sucrose. The vertical axis Figure 11. Viability of SC-1 R780 fibroblasts in the presence of glycerol or sucrose. The vertical axis denotes the percentage of viable cells, and the horizontal axis shows fold dilution of stock solutions Figure 11. Viability of SC-1 R780 fibroblasts in the presence of glycerol or sucrose. The vertical axis denotes the percentage of viable cells, and the horizontal axis shows fold dilution of stock solutions of glycerol (75%) and sucrose (58%). denotes the percentage of viable cells, and the horizontal axis shows fold dilution of stock solutions of glycerol (75%) and sucrose (58%). of glycerol (75%) and sucrose (58%). These findings indicate that the presence of glycerol in the culture medium, even at These findings indicate that the presence of glycerol in the culture medium, even at low concentrations, has an adverse effect on cells. Therefore, the MLNC preparations ob- These findings indicate that the presence of glycerol in the culture medium, even low concentrations, has an adverse effect on cells. Therefore, the MLNC preparations ob- tained using the purification method involving fractionation of samples in glycerol is not at low concentrations, has an adverse effect on cells. Therefore, the MLNC preparations tained using the purification method involving fractionation of samples in glycerol is not suitable for research on cultured cells. We present the results of this study to draw the obtained using the purification method involving fractionation of samples in glycerol is suitable for research on cultured cells. We present the results of this study to draw the Nanomaterials 2021, 11, 2775 15 of 16 not suitable for research on cultured cells. We present the results of this study to draw the readers’ attention to the necessity of comprehensive characterization before the approval of methods intended for nanobiotechnology and nanomedicine. 4. Conclusions Lipid-coated particles that serve as a carrier of siRNA are the subject of numerous studies. Several years ago, we published the proof of principle for the construction of an AuNP-based MLNC that efficiently delivers siRNA into the cell [15]. Nonetheless, we were not satisfied with the quality of the obtained nanocomposites, and thus, here we found some ways to improve it in comparison with the original version. In this work, we demonstrated that even seemingly insignificant modifications of the steps of the MLNC fabrication affect end product quality. For example, the optimal reaction mixture for obtaining the core nanoparticles contains 0.1 mM MgSO and 5 mM NaCl; lipid film synthesis at 12 mmHg without thermostatting improves the quality of the forming MLNCs, as does the assembly of MLNCs in 1 mM phosphate buffer. Having optimized all the steps of MLNC fabrication, we noticed that 15 min centrifugation at 2000 g in 58% sucrose yields a fraction containing 40% of target MLNCs, i.e., a doubled proportion of these nanoconstructs as compared to the end product of the original procedure [15]. We think that this study can help researchers who design nanoconstructs based on metal nanoparticles coated with a lipid envelope. 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Journal

Nanomaterials – Multidisciplinary Digital Publishing Institute

Published: Oct 20, 2021

Keywords: gold nanoparticles; siRNA; noncovalent adsorption; lipid enveloping; multilayer nanoconstructs for siRNA preparation and purification

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