Citation: Szabó, I.; Yousef, M.; Soltész, D.; Bató, C.; Mez ˝ o, G.; Bánóczi, Z. Redesigning of Cell-Penetrating Peptides to Improve Their Efficacy as a Drug Delivery System. Pharmaceutics 2022, 14, 907. https://doi.org/10.3390/ pharmaceutics14050907 Academic Editors: João Augusto Oshiro Júnior and Arnobio A. da Silva-Junior Received: 18 March 2022 Accepted: 13 April 2022 Published: 21 April 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 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 Review Redesigning of Cell-Penetrating Peptides to Improve Their Efficacy as a Drug Delivery System Ildikó Szabó 1, *, Mo’ath Yousef 2 ,Dóra Soltész 2 , Csaba Bató 2 ,Gábor Mez ˝ o 1,2 and ZoltánBánóczi 2, * 1 MTA-ELTE Research Group of Peptide Chemistry, Eötvös Loránd Research Network (ELKH), Eötvös Loránd University, 1117 Budapest, Hungary; gabor.mezo@ttk.elte.hu 2 Department of Organic Chemistry, Institute of Chemistry, Eötvös Loránd University, 1117 Budapest, Hungary; yousefmoath@gmail.com (M.Y.); soltesz.dora6@gmail.com (D.S.); bs.csabi@gmail.com (C.B.) * Correspondence: ildiko.szabo@ttk.elte.hu (I.S.); zoltan.banoczi@ttk.elte.hu (Z.B.) Abstract: Cell-penetrating peptides (CPP) are promising tools for the transport of a broad range of compounds into cells. Since the discovery of the first members of this peptide family, many other pep- tides have been identified; nowadays, dozens of these peptides are known. These peptides sometimes have very different chemical–physical properties, but they have similar drawbacks; e.g., non-specific internalization, fast elimination from the body, intracellular/vesicular entrapment. Although our knowledge regarding the mechanism and structure–activity relationship of internalization is growing, the prediction and design of the cell-penetrating properties are challenging. In this review, we focus on the different modifications of well-known CPPs to avoid their drawbacks, as well as how these modifications may increase their internalization and/or change the mechanism of penetration. Keywords: cell-penetrating peptide; chemical modification; peptide conjugate; drug delivery; internalization 1. Introduction The story of cell-penetrating peptides started with the discovery that two short pep- tides, which are part of large proteins, can penetrate the cell membrane. These first two cell-penetrating peptides were Tat [1,2] and penetratin [3]. These early results opened the gate to dozens of peptides with cell-penetrating properties [4,5]. These peptides were used to deliver a wide range of biologically active molecules into cells [6–9]. Although many peptides were described as CPPs, they all have some common properties that make them able to penetrate cells. Usually, they are positively charged due to their high content of basic amino acids (especially arginine and lysine); they are, at maximum, 30-amino acid-length peptide sequences; and sometimes they have well-defined secondary structures when they interact with cell membranes [10,11]. Despite their high number and diverse structure, designing a CPP is challenging, and the cell-penetrating property of a peptide is unpredictable. Some peptides have additional advantages; e.g., penetration through the blood–brain barrier [12,13], tumor-homing [14,15], and antimicrobial activity [16,17]. Although they have often been used successfully to deliver cargos into cells, they have a number of limitations, such as non-specific internalization, endosomal entrapment of cargo, limited stability, and fast elimination from the body. The intent to avoid these drawbacks, and to improve CPPs for in vivo applications, has induced much research, and has resulted in the development of new approaches in this field. The increasing number of chemical modifications highlights that, similar to the CPP peptides, there is no generally applicable solution, and only the main direction can be seen. In this review, we summarize the results of these attempts; in particular, the chemical modifications that allow fast and easy improvement of CPPs. Pharmaceutics 2022, 14, 907. https://doi.org/10.3390/pharmaceutics14050907 https://www.mdpi.com/journal/pharmaceutics