Citation: Onishchenko, N.R.; Moskovtsev, A.A.; Kobanenko, M.K.; Tretiakova, D.S.; Alekseeva, A.S.; Kolesov, D.V.; Mikryukova, A.A.; Boldyrev, I.A.; Kapkaeva, M.R.; Shcheglovitova, O.N.; et al. Protein Corona Attenuates the Targeting of Antitumor Sialyl Lewis X-Decorated Liposomes to Vascular Endothelial Cells under Flow Conditions. Pharmaceutics 2023, 15, 1754. https://doi.org/10.3390/ pharmaceutics15061754 Academic Editors: Franco Dosio and Xiaowei Zeng Received: 17 April 2023 Revised: 23 May 2023 Accepted: 12 June 2023 Published: 16 June 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). pharmaceutics Article Protein Corona Attenuates the Targeting of Antitumor Sialyl Lewis X-Decorated Liposomes to Vascular Endothelial Cells under Flow Conditions Natalia R. Onishchenko 1,†,‡ , Alexey A. Moskovtsev 2,† , Maria K. Kobanenko 1 , Daria S. Tretiakova 1 , Anna S. Alekseeva 1 , Dmitry V. Kolesov 2 , Anna A. Mikryukova 2 , Ivan A. Boldyrev 1 , Marina R. Kapkaeva 3 , Olga N. Shcheglovitova 3 , Nicolai V. Bovin 1 , Aslan A. Kubatiev 2 , Olga V. Tikhonova 4 and Elena L. Vodovozova 1, * 1 Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia; natalia.r.onishchenko@gmail.com (N.R.O.) 2 Institute of General Pathology and Pathophysiology, Russian Academy of Sciences, ul. Baltiyskaya 8, 125315 Moscow, Russia 3 N.F. Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Healthcare of the Russian Federation, ul. Gamaleya 18, 123098 Moscow, Russia 4 Institute of Biomedical Chemistry, ul. Pogodinskaya 10, 119121 Moscow, Russia * Correspondence: elvod.ibch@yandex.ru † These authors contributed equally to this work. ‡ Current address: Center for Soft and Living Matter, Institute for Basic Science, UNIST-gil 50 bldg. 103, Ulsan 44919, Republic of Korea. Abstract: Previously, we showed in the human umbilical vein endothelial cells (HUVECs) model that a liposome formulation of melphalan lipophilic prodrug (MlphDG) decorated with selectin ligand tetrasaccharide Sialyl Lewis X (SiaLe X ) undergoes specific uptake by activated cells and in an in vivo tumor model causes a severe antivascular effect. Here, we cultured HUVECs in a microfluidic chip and then applied the liposome formulations to study their interactions with the cells in situ under hydrodynamic conditions close to capillary blood flow using confocal fluorescent microscopy. The incorporation of 5 to 10% SiaLe X conjugate in the bilayer of MlphDG liposomes increased their consumption exclusively by activated endotheliocytes. The increase of serum concentration from 20 to 100% in the flow resulted in lower liposome uptake by the cells. To elucidate the possible roles of plasma proteins in the liposome–cell interactions, liposome protein coronas were isolated and analyzed by shotgun proteomics and immunoblotting of selected proteins. Proteomic analysis showed that a gradual increase in SiaLe X content correlated with the overall enrichment of the liposome-associated proteins with several apolipoproteins, including the most positively charged one, ApoC1, and serum amyloid A4, associated with inflammation, on the one hand, and a decrease in the content of bound immunoglobulins, on the other. The article discusses the potential interference of the proteins in the binding of liposomes to selectins of endothelial cells. Keywords: nanosized liposomes; lipophilic prodrug; melphalan; Sialyl Lewis X; endothelial cells; microfluidics; proteome 1. Introduction Today, liposomal formulations of anticancer drugs are used to treat a whole number of tumor diseases in clinics [1–4]. Parenteral administration of drug-loaded nanosized lipo- somes permits the alleviation of systemic toxicity of small-molecule antitumorials through lower concentration of the free drug in blood and tendency of nanoparticles to accumulate in tumors’ impaired vasculature (enhanced permeability and retention effect, EPR) [5,6]. Yet ligand-mediated targeting of tumors continues to be a challenge owing, first of all, to the vascular and interstitial barriers [1,7,8]. Further, spatial and temporal heterogeneity in Pharmaceutics 2023, 15, 1754. https://doi.org/10.3390/pharmaceutics15061754 https://www.mdpi.com/journal/pharmaceutics