**Anna S. Pavlova, Ilya S. Dovydenko, Maxim S. Kupryushkin, Alina E. Grigor'eva, Inna A. Pyshnaya and Dmitrii V. Pyshnyi \***

Institute of Chemical Biology and Fundamental Medicine SB RAS, 630090 Novosibirsk, Russia; pavlova@niboch.nsc.ru (A.S.P.); dovydenko\_il@niboch.nsc.ru (I.S.D.); kuprummax@niboch.nsc.ru (M.S.K.); grigoryeva@niboch.nsc.ru (A.E.G.); pyshnaya@niboch.nsc.ru (I.A.P.)

**\*** Correspondence: pyshnyi@niboch.nsc.ru; Tel.: +7-383-363-5151

Received: 31 August 2020; Accepted: 25 September 2020; Published: 29 September 2020

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

**Keywords:** lipid conjugates; amphiphilic oligonucleotides; self-assembly; phosphoryl guanidines; nucleic acid delivery
