International Journal of Molecular Sciences Article Hybrid Cyclobutane/Proline-Containing Peptidomimetics: The Conformational Constraint Influences Their Cell-Penetration Ability Ona Illa 1, * , Jimena Ospina 1 , José-Emilio Sánchez-Aparicio 1 , Ximena Pulido 2,3,4 , María Ángeles Abengozar 5 , Nerea Gaztelumendi 6 , Daniel Carbajo 7 , Carme Nogués 6 , Luis Rivas 5 , Jean-Didier Maréchal 1 , Miriam Royo 3,7, * and Rosa M. Ortuño 1, * 1 Departament de Química, Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Spain; jimenabcn@gmail.com (J.O.); joseemilio.sanchez@uab.cat (J.-E.S.-A.); jeandidier.marechal@uab.cat (J.-D.M.) 2 Institut de Recerca Biomèdica, c/Baldiri Reixac 10, 08028 Barcelona, Spain; xpulido@ut.edu.co 3 Centro de Investigación Biomédica en Red Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), c/ Jordi Girona 18–26, 08034 Barcelona, Spain 4 Departamento de Química, Universidad del Tolima, Santa Helena Parte Alta, Ibagué 730006299, Tolima, Colombia 5 Centro de Investigaciones Biológicas Margarita Salas, c/ Ramiro de Maeztu 9, CSIC, 28040 Madrid, Spain; marabi66@hotmail.com (M.Á.A.); luis.rivas@cib.csic.es (L.R.) 6 Departament de Biologia Cellular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona,  08193 Cerdanyola del Vallès, Spain; negazte@gmail.com (N.G.); carme.nogues@uab.cat (C.N.)  7 Institut de Química Avançada de Catalunya (IQAC-CSIC), c/ Jordi Girona, 18-26, 08034 Barcelona, Spain; Citation: Illa, O.; Ospina, J.; daniel.carbajo@iqac.csic.es * Correspondence: ona.illa@uab.cat (O.I.); miriam.royo@iqac.csic.es (M.R.); rosa.ortuno@uab.cat (R.M.O.) Sánchez-Aparicio, J.-E.; Pulido, X.; Abengozar, M.Á.; Gaztelumendi, N.; Carbajo, D.; Nogués, C.; Rivas, L.; Abstract: A new family of hybrid β,γ-peptidomimetics consisting of a repetitive unit formed by a Maréchal, J.-D.; et al. Hybrid chiral cyclobutane-containing trans-β-amino acid plus a Nα -functionalized trans-γ-amino-L-proline Cyclobutane/Proline-Containing joined in alternation were synthesized and evaluated as cell penetrating peptides (CPP). They Peptidomimetics: The Conformational lack toxicity on the human tumoral cell line HeLa, with an almost negligible cell uptake. The Constraint Influences Their dodecapeptide showed a substantial microbicidal activity on Leishmania parasites at 50 µM but with a Cell-Penetration Ability. Int. J. Mol. Sci. modest intracellular accumulation. Their previously published γ,γ-homologues, with a cyclobutane 2021, 22, 5092. https://doi.org/ γ-amino acid, showed a well-defined secondary structure with an average inter-guanidinium distance 10.3390/ijms22105092 of 8–10 Å, a higher leishmanicidal activity as well as a significant intracellular accumulation. The presence of a very rigid cyclobutane β-amino acid in the peptide backbone precludes the acquisition Academic Editor: Andrew Clayton of a defined conformation suitable for their cell uptake ability. Our results unveiled the preorganized charge-display as a relevant parameter, additional to the separation among the charged groups as Received: 19 March 2021 Accepted: 7 May 2021 previously described. The data herein reinforce the relevance of these descriptors in the design of Published: 11 May 2021 CPPs with improved properties. Publisher’s Note: MDPI stays neutral Keywords: peptidomimetics; cell penetrating peptides; conformational bias; rigidity; charge- with regard to jurisdictional claims in preorganization published maps and institutional affil- iations. 1. Introduction Cell penetrating peptides (CPPs) [1–3] are potential carriers for drug delivery systems Copyright: © 2021 by the authors. (DDS). They usually consist of peptide oligomers that convey the molecular payload into Licensee MDPI, Basel, Switzerland. the intracellular space in the absence of a cognate transporter/receptor. One of their major This article is an open access article drawbacks is their poor cellular specificity. This can be partially overcome by their local distributed under the terms and activation through the degradation of an inhibitory fragment carried out by specific pro- conditions of the Creative Commons teinases, under environmental conditions (pH, hypoxia, etc.) or by the inclusion into their Attribution (CC BY) license (https:// sequence of recognition motifs for a specific receptor located in the targeted cell [4]. A step creativecommons.org/licenses/by/ ahead is the delivery of a cargo molecule into a certain organelle by inclusion of a particular 4.0/). Int. J. Mol. Sci. 2021, 22, 5092. https://doi.org/10.3390/ijms22105092 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 5092 2 of 18 import sequence or by physicochemical characteristics of a given peptide [5]. The pharma- cological implementation of CPPs as DDS [6–8] is supported by their versatility to transport cargo molecules through covalent and non-covalent interactions, high bioavailability, fine tuning of their solubility, and by the feasibility to design multifunctional DDS [9]. As such, CPPs were implemented as a new tool in the therapeutics of a variety of diseases, with a special relevance in cancer [10]. CPP engineering aims to achieve an optimal biological performance by ensuring an adequate cell uptake and endosomal escape, with minimal toxicity, as well as to expand the half-life of the peptide under biological environments. This last property mostly relies on the improvement of its proteolytic resistance. To this end, the inclusion in their sequence of non-natural or stereochemically modified amino acids, as well as of peptide bond surrogates have been described [11]. The biological activity and selectivity of proteins and peptides is highly influenced by the conformational restriction of their polypeptide backbone. A case in point is the neuropeptide Y (NPY), which controls central and peripheral processes by the activation of the G protein coupled receptors Yx R. Meanwhile, the parental peptide shows promiscuous binding among different receptors of this family, analogues of its C-terminal fragment containing cyclobutane or cyclopentane β-amino acids displayed exclusively binding to Y4 R [12]. A brand-new example is the incorporation of six proline residues in the spike glycoprotein of coronavirus virions with improved recombinant expression, and tolerance heat stress, thus allowing their storage at room temperature [13]. Otherwise, local constriction in biologically active peptides is another useful tool to improve their activity. For instance, the modification of the hydrazide bridge in biphalin, an opioid octapeptide, significantly enhances its analgesic properties [14]. This is the case, for instance, of biphalin analog AM94, where the native hydrazine linker has been replaced by a more constrained and less planar piperazine ring. AM94 displayed a higher potency than biphalin and a comparable effect with morphine [15]. Another example is provided by the incorporation of side-chain constrained α- or β-substituted amino acids into bioactive peptide ligands as key pharmacophore residues. It has proven to be a powerful method for understanding ligand–receptors binding interaction and in peptidomimetics design [16]. Conformational constraints were also introduced to stabilize the secondary structure of CPPs, sometimes associated with a better cell uptake [11]. For cationic CPPs, uptake and toxicity are highly dependent on the number and spatial distribution of positive charges throughout their sequence. With these two goals in mind, diverse CPPs were designed by combining in a single unit two cyclic amino acids, mainly proline and γ-aminoproline [17,18], helical peptide foldamers [19–21], and cyclic peptide backbones [22–25]. In previous works, we reported the biological performance as CPP of hybrid γ,γ- peptides formed by repetition of a dimeric unit constituted by a protected derivative of either cyclobutane γ-amino acids (γ-CBAA), 1 or 2 combined with cis-γ-amino-L-proline, 3 (Figure 1). High-resolution NMR spectra of these compounds showed very rigid and com- pact structures due to the intra- and inter-residue hydrogen-bonded ring formation [26]. For these peptides, the uptake by tumoral human cell line HeLa increased with its length, while the stereochemistry of the γ-CBAA was scarcely relevant. A good polar- hydrophobicity balance was achieved by the alternation of the guanidinium groups and the hydrophobicity of the (gem-dimethyl) cyclobutane ring [26,27]. These cyclobutane- containing CPPs showed a lower toxicity than those made exclusively of guanidinylated γ-amino proline residues though preserving a similar cell uptake [28], likely due to their halved number of guanidinium groups compared with the γ-aminoproline peptides of the same length. Later, we synthesized the oligomers (from 8 to 14 residues) formed by 1 and cis- or trans-γ-amino-L-proline, 3 and 4, as the bases for γ-CC and γ-CT, respectively (Figure 1). These peptides bear guanidinium groups attached to the backbone through a flexible spacer. They were conjugated to carboxyfluorescein (CF) used as a fluorophore and were evaluated as CPPs and as selective vectors for anti-Leishmania DDS. These peptides and Int. Int.J.J.Mol. Mol.Sci. Sci.2021, 2021,22, 22,xxFOR FORPEER PEERREVIEW REVIEW 33ofof19 19 Int. J. Mol. Sci. 2021, 22, 5092 3 of 18 spacer. spacer.They Theywerewereconjugated conjugatedtotocarboxyfluorescein carboxyfluorescein(CF) (CF)used usedasasaafluorophore fluorophoreandandwere were evaluated evaluated as CPPs and as selective vectors for anti-Leishmania DDS. These peptidesand as CPPs and as selective vectors for anti-Leishmania DDS. These peptides and their theirrespective respectiveCF-conjugates respective CF-conjugateslacked CF-conjugates lacked cytotoxicity lacked cytotoxicity cytotoxicity on HeLa onon cells HeLa HeLa with cells cells with aamoderate with cell-up- a moderate moderate cell- cell-up- take takeon uptakeonthem. on In Incontrast, them. them. both In contrast, contrast, γ-CC bothboth and andγ-CT γ-CCγ-CC tetradecamers and γ-CT γ-CT were weremicrobicidal tetradecamers tetradecamers were on onLeish- microbicidal microbicidal on Leish- mania maniabeyond Leishmania beyond 25 25µµM, beyond 25with M, withawith µM, asignificant intracellular a significant significant accumulation intracellular intracellular ininthis accumulation accumulation inprotozoan this par- this protozoan protozoan par- asite asite[29]. parasite [29].[29]. Figure Figure1.1. Figure 1.Monomers, Monomers,typetypeofofpeptides, peptides,and andcarboxyfluorescein and carboxyfluorescein(CF) carboxyfluorescein (CF)conjugates (CF) conjugatesinvestigated conjugates investigatedinin investigated inour our our earlier earlierstudies studies[27,28,30]. [27,28,30]. earlier studies [26,27,29]. These Thesepeptides peptidesadopted adoptedaadefined adopted definedconformational defined conformationalpreference conformational preferencein preference insolution, in solution,as solution, asassessed as assessed assessed by bycircular circular dichroism dichroism (CD) (CD) spectroscopy. spectroscopy. In Inaddition, addition, well-defined well-defined circular dichroism (CD) spectroscopy. In addition, well-defined conformations conformations conformations for forboth both for tetradecamers tetradecamers both were tetradecamers inferred were inferred by molecular by molecular were inferred dynamics dynamics by molecular (MD) simulations, (MD) simulations, dynamics driven by driven by (MD) simulations, the for- the for- driven by mation mation the ofofintra- formation and intra-of and long-range long-range intra- inter-residue inter-residue and long-range hydrogen hydrogenbonds. inter-residue bonds.As hydrogen Asananexample, example, bonds. representa- representa- As an example, tive tiveconformations representative conformations for forγ-CT γ-CTand conformations for for and forCF-γ-CT γ-CT and forare CF-γ-CT displayed CF-γ-CT are ininFigure Figure2. are displayed displayed 2.To in Tonote, Figure 2.the note, To con- the note, con- formational the formational restrictions conformational restrictions for forγ,γ-peptides restrictions are aremuch for γ,γ-peptides γ,γ-peptides higher are much much than higher higher for forpeptides thanthan made for peptides peptides made madeofofα-of α- amino acids. Moreover, an important fact is the location of polar guanidinium amino acids. Moreover, an important fact is the location of polar guanidinium groups at α-amino acids. Moreover, an important fact is the location of polar guanidinium groups groups at at the theperiphery peripheryof ofthe thepeptide the peptidefacing peptide facingthe facing thesolvent. the solvent.This solvent. Thiswill willimprove improvetheir improve theirsolubility their solubilityand solubility andthe and the the interaction interactionwith withthe thenegatively negativelycharged negatively chargedpeptidoglycans charged peptidoglycansof peptidoglycans ofthe theextracellular the extracellularmatrix extracellular matrixof matrix ofthe of the the targeted targetedcell targeted cell[28]. cell [29]. [28]. Figure 2. Representative MD conformations for (a) peptide γ-CT, with inter-residue interactions Figure Figure2.2.Representative RepresentativeMD MDconformations conformations for for(a) (a)peptide peptideγ-CT, with inter-residue interactions are marked; and (b) CF-γ-CT. The green ribbon stands outγ-CT, with for the inter-residue peptide scaffold.interactions CF moiety is are aremarked; marked;andand(b) (b)CF-γ-CT. CF-γ-CT.The Thegreen greenribbon ribbonstands standsout outfor forthe thepeptide peptidescaffold. scaffold.CF CFmoiety moietyisis representedin represented in magenta. represented inmagenta. magenta. To pursue a better definition on the role of conformational restriction of cyclobutane- To Topursue pursueaabetter betterdefinition definition on onthe role theto role ofofconformational conformational restriction restriction ofofcyclobutane- cyclobutane- containing peptidomimetics with respect their cell penetrating activity, we synthetized new containing containing peptidomimetics peptidomimetics with withrespect respect tototheir their cell cellpenetrating penetrating activity, activity,we we synthetized synthetized hybrid β,γ-oligomers that incorporate the highly constrained (1S,2S)-2-aminocyclobutane-1- carboxylic acid, 5, (Figure 3). In previous studies, it was observed that the inclusion of 5 in short oligomers (2–12 residues) led to well defined helical folding in solution prompted by the Int. J.J. Mol. Int. Mol. Sci. Sci. 2021, 2021, 22, 22, xx FOR FOR PEER PEER REVIEW REVIEW 44 of of 19 19 Int. J. Mol. Sci. 2021, 22, 5092 4 of 18 new hybrid new hybrid β,γ-oligomers β,γ-oligomers thatthat incorporate incorporate the the highly highly constrained constrained (1S,2S)-2-aminocyclo- (1S,2S)-2-aminocyclo- butane-1-carboxylic acid, 5, (Figure 3). In previous studies, it was observed butane-1-carboxylic acid, 5, (Figure 3). In previous studies, it was observed that that the the inclu- inclu- sion of 5 in short oligomers (2–12 residues) led to well defined helical folding sion of 5 in short oligomers (2–12 residues) led to well defined helical folding in solution in solution promptedof formation prompted byeight-membered by the formation the formation of ofhydrogen-bonded eight-memberedrings eight-membered hydrogen-bonded rings involving i andrings hydrogen-bonded involving i + 1involving ii and and ii residues [30–32]. + 1 Thisresidues [30–32]. type of hydrogen + 1 residues This type bonding [30–32]. This typewas of hydrogen bonding also foundbonding of hydrogen was in preliminaryalso found studies was also in preliminary foundoninhybrid studies β,γ-oligomers preliminary studies on hybrid (2–4 on hybrid β,γ-oligomers residues) containing N β,γ-oligomers (2–4 (2–4 residues) α -Boc containing aminoproline residues) Nαα-Boc -Boc(Boc derivatives containing N aminoproline derivatives (Boc = tert-butoxycarbonyl) aminoproline derivatives (Boc [33] = tert-butoxycarbonyl) (Figure 3). [33] (Figure = tert-butoxycarbonyl) [33] (Figure 3). 3). Figure 3. Figure 3. Eight-membered Eight-membered hydrogen-bonded hydrogen-bonded rings rings in in oligomers oligomers containing containing the the cyclobutane cyclobutane amino amino amino acid 5. acid 5. acid 5. In order In order toto get get aa better better insight insight into into the the relevance relevance of of conformational conformational constraints conformational constraints in constraints in the in the the CPP scaffolds CPP scaffolds and and their their biological biological performance, performance, peptides peptides 6–11 6–11 (Figure (Figure 4) 4) were were compared compared 4) were compared with the with the homonymous homonymous oligomers homonymousoligomers containing oligomerscontaining the containingthe γ-amino the γ-amino γ-amino acid 11 (Figure acid acid (Figure 1 (Figure1). Their 1). Their cell pen- 1). Their cell pen- cell etrating penetratingactivity was activity etrating activity waswasassayed assayed assayed in two in in two two different biological differentbiological different models, biologicalmodels, HeLa models,HeLa cells HeLacells and cells and the and the twotwo two major forms of Leishmania, major forms of Leishmania, a human protozoan Leishmania, a human protozoan parasite. protozoan parasite. In addition, MD simulations parasite. In addition, MD simulations were simulations were were carried out carried out to to predict predict the predict the representative the representative conformations. representative conformations. Altogether, conformations. Altogether, thethe importance importance of importance of aaa specific topology specific topology of of the the guanidium guanidium group group on on CPPs CPPs incorporating incorporating β,γ- β,γ- or or topology of the guanidium group on CPPs incorporating β,γ- or γ,γ- amino acids γ,γ- γ,γ- amino amino acids acids was verified. was was verified. verified. Figure 4. Figure Figure 4. Hybrid 4. Hybrid β,γ-peptides Hybrid β,γ-peptides and β,γ-peptides and their their CF-conjugates CF-conjugates involved involved in in this work. this work. work. 2. Results 2. Results Results andand Discussion and Discussion Discussion 2. 2.1. Synthesis of the Peptides 2.1. Synthesis 2.1. Synthesis of of the the Peptides Peptides To analyze the role of peptide length on the cell uptake, a series of hybrid β,γ-peptides To analyze 6–11 To analyze (Figure 4, n =the the role role 4–6) of synthesized. of was peptide length peptide length onwere on They the cell the cell uptake, uptake, formed aa series by the series of hybrid of repetitionhybrid β,γ-pep- β,γ-pep- of a dipeptide tides 6–11 tides 6–11 motif (Figure made(Figure up by 4, 4, n = 4–6) was synthesized. n = 4–6) was synthesized. (1S,2S)-β-CBAA, They 5 [34] and They were formed were formed trans-γ-amino- by the repetition by the 4.repetition L -proline, of aa of All of them dipeptide dipeptide were motifusing motif prepared madestandard made up by up by (1S,2S)-β-CBAA, (1S,2S)-β-CBAA, 55 [34] protocols of solid [34] andsynthesis and phase trans-γ-amino- trans-γ-amino- L methods -proline, L-proline, (SPPS), 4.either 4. All of All of them them in were were their prepared freeprepared N-terminus using using form standard standard protocols (6–8) orprotocols of solid phase of solidasphase with CF (9–11), synthesis synthesis detailed methods methods in Section (SPPS), 4 and(SPPS), in the either in either in their their free Supplementary free N-terminus N-terminus Materials form (6–8) form (6–8) or (Supplementary orTables with CF with CF (9–11), as (9–11), S1–S3). as detailed detailed inin Section Section 44 and and inin the Supplementary the Supplementary Materials (Supplementary Materials (Supplementary TAT48–57 (transactivator Tables of transcription,Tables S1–S3). TAT) S1–S3). [35–37] was also synthesized as a TAT48–57 TAT reference 48–57 (transactivator of transcription, TAT) [35–37] was also synthesized as a ref- CPP, (transactivator either with aoffree transcription, TAT) [35–37] wasN-terminus or carboxyfluoresceinated also synthesized as a ref- as previously erence erence CPP, CPP, described [29]. either either with with a a free free or or carboxyfluoresceinated carboxyfluoresceinated N-terminus N-terminus as as previously previously de- de- scribed [29]. scribed [29]. 2.2. Cytotoxicity and Cellular Uptake in HeLa Cells The cellular viability of peptides 6–11 was determined by the reduction of MTT (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [38] after 24 h of cell Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 of 19 2.2. Cytotoxicity and Cellular Uptake in HeLa Cells Int. J. Mol. Sci. 2021, 22, 5092 The cellular viability of peptides 6–11 5 of 18 2.2. Cytotoxicity and Cellular Uptake in HeLa Cellswas determined by the reduction of MTT (3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay [38] after 24 h of cell The cellular viability of peptides 6–11 was determined by the reduction of MTT (3- incubation with the respective oligomer. The measure (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)ofassay the formazan formed [38] after 24 h of cellfrom the reduction incubation incubation ofwith MTT with byrespective the the mitochondrial respective dehydrogenases oligomer. oligomer. The The measure measure ofis of directly the the related formazan formazan to the formed formed cellular from from the via- the reduction bility of MTT by mitochondrial dehydrogenases is directly related to reduction of MTT by mitochondrial dehydrogenases is directly related to the cellular via- over (Figure 5). Even at the highest concentration assayed (50 µ M), the viabilitycellular was 90%viability for bility (Figure this (Figure set 5).of 5). Even Even peptides. at theat the highest Peptide highest concentration toxicity assayed wasassayed concentration not (50(50 dependent µM), µ M), viability either onwas viability was the over number over of 90% for guanidinium this 90% for this set set of groups peptides. Peptide in thePeptide of peptides. sequence toxicity or on toxicity was wasthe not dependent either notcarboxyfluoresceinationon the dependent either on the numbernumber of the of of terminal guanidinium guanidinium groups inin the sequence orononthethe carboxyfluoresceinationofofthe theterminal terminal amino group, groups in agreement the sequence with the or previous carboxyfluoresceination data on their γ,γ-counterparts, γ-CC and γ- amino amino group, group, in in agreement agreement withwith the the previous previous data data on on their their γ,γ-counterparts, γ,γ-counterparts, γ-CC γ-CC and and γ- CTγ-CT (Figure 1) [29]. (Figure 1) [29]. CT (Figure 1) [29]. Figure 5. Cytotoxicity of peptides 6–11 on HeLa cells. Peptides with (a) Free terminal amino group; (b) terminal amino Figure 5. Cytotoxicity of peptides 6–11 on HeLa cells. Peptides with (a) Free terminal amino group; (b) terminal amino Figure 5. Cytotoxicity of peptides 6–11 on HeLa cells. Peptides with (a) Free terminal amino group; (b) terminal amino group group conjugated conjugated with CF. Cells were incubated with the respective peptide forfor 2424h and their viability assayed bybyMTT group conjugated reduction. Cell withwith CF. viability CF. was Cells Cells were were expressed as incubated incubated the with with percentage the the of respective respective MTT peptide reductionpeptide with for 24 respect h and their h control to and viability their assayed viability cells ± SD. assayed Experiments MTT by MTT reduction. reduction. Cell Cell viability viability was was expressed expressed asas thethe percentage percentage of of MTT MTT reduction reduction with with respect respectto control to cells control ± SD. cells ± Experiments SD. Experiments were repeated thrice independently. Statistical significance respect to untreated control (p ≤ 0.05, (*); p ≤ 0.001, (***)). were were repeated repeated thrice thrice independently.Statistical independently. Statisticalsignificance significance respect respecttotountreated untreatedcontrol (p ≤(p0.05, control ≤ 0.05, ≤ 0.001, (*); p(*); (***)).(***)). p ≤ 0.001, Their Their cell internalization cell internalization ability inin ability HeLa HeLacells was cells wasassessed assessedbybyflow flowcytometry. cytometry. ToTothis this end, Their the cell internalization cell-associated ability fluorescence in was HeLa cells quantified was after assessed incubation end, the cell-associated fluorescence was quantified after incubation with peptides 9–11 by flow with cytometry. peptides 9–11 To this end, at at the 1010 and cell-associated and 2525µM µM and and fluorescence compared compared with withthatwas quantified obtained that obtained with with after CF-TAT incubation CF-TAT asasa astandardwithreference standard peptides 9–11 reference at(Figure 10 and (Figure6).6). 25Results µ M and Results showed a modest compared showed cell-uptake with a modest forfor that obtained cell-uptake allall peptides, with being CF-TAT peptides, being the dodecamer asthe standardthe a dodecamer reference the peptide (Figure peptide with 6).with the Results highest value. showedvalue. the highest a modest cell-uptake for all peptides, being the dodecamer the peptide with the highest value. Figure Figure 6. 6. Cellular Cellular internalizationofofcarboxyfluoresceinated internalization carboxyfluoresceinatedpeptides peptides9–11 normalized with 9–11normalized with respect respect to to CF-TAT CF-TAT(100%). (100%).HeLa HeLacells cellswere wereincubated incubatedwith withthe therespective respectivepeptide peptideatat1010µM μM(empty (empty column), col- or 25 µM umn), or (black 25 μMcolumn) for 2 h at (black column) for372◦hC,atand the and 37 °C, levelthe of fluorescence associated level of fluorescence with the with associated cells assessed the cells by assessed Figure flow by flow cytometry 6. Cellular (λcytometry EXC = 488 nm internalization (λEXC ofand= 488 λEMnm andnm). = 530 λEM =Results carboxyfluoresceinated 530 nm). Results were wereas expressed peptides 9–11 expressed normalized as with mean ± SD. Threerespect mean ± SD. independent Three independent experiments experiments werewere carried were carried out. Statistical out. Statistical significance respect to ≤ 0.001, CF- to CF-TAT (100%). HeLa cells incubated withsignificance respect the respective to CF-TAT peptide at 10(pμM (empty(***)). col- TAT (p ≤ 0.001, (***)). umn), or 25 μM (black column) for 2 h at 37 °C, and the level of fluorescence associated with the The intracellular uptake of the peptides was assessed by confocal microscopy. Figure 7 cells assessed by flow cytometry (λEXC = 488 nm and λEM = 530 nm). Results were expressed as shows HeLa cells after 24 h incubation with the dodecamer 11 at 10 µM. CellMask deep red mean ± SD. Three independent experiments were carried out. Statistical significance respect to CF- and Hoechst were used to stain the plasma membrane (red), and DNA (blue), respectively. TAT (p ≤ 0.001, (***)). The CF-peptide accounts for the green fluorescence. Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 of 19 The intracellular uptake of the peptides was assessed by confocal microscopy. Figure Int. J. Mol. Sci. 2021, 22, 5092 7 shows HeLa cells after 24 h incubation with the dodecamer 11 at 10 µ M. CellMask6 deep of 18 red and Hoechst were used to stain the plasma membrane (red), and DNA (blue), respec- tively. The CF-peptide accounts for the green fluorescence. Plasma membrane Peptide 11 Nuclei Merge Figure 7. Figure 7. Intracellular Intracellular location location of ofthe thefluorescent fluorescentpeptide peptide11. 11.Confocal Confocalmicroscopy microscopy of of HeLa HeLacells cells incubated with the carboxyfluoresceinated dodecamer 11 (incubation conditions: 2 h, incubated with the carboxyfluoresceinated dodecamer 11 (incubation conditions: 2 h, 37 C, 10 37◦ °C, 10µM) μM) (green fluorescence λEXC = 488 nm; λEM = 510 nm). Plasma membrane was additionally stained (green fluorescence λEXC = 488 nm; λEM = 510 nm). Plasma membrane was additionally stained with with CellMask, deep red (red fluorescence, λEXC = 658 nm; λEM = 690 nm), and nucleic acids with CellMask, deep red (red fluorescence, λEXC = 658 nm; λEM = 690 nm), and nucleic acids with Hoechst Hoechst (blue fluorescence, λEXC = 405 nm; λEM = 460 nm). Magnification bar: 10 µ m. (blue fluorescence, λEXC = 405 nm; λEM = 460 nm). Magnification bar: 10 µm. 2.3. Uptake, 2.3. Uptake,Microbicidal MicrobicidalActivity, Activity,and andIntracellular IntracellularLocation LocationofofPeptides PeptidesononLeishmania Parasites Leishmania Parasites Once the lack of cytotoxicity and poor uptake of the peptides respect to TAT on HeLa Once cells was the lack oftheir evidenced, cytotoxicity performance and poor uptake of the on Leishmania peptides parasites wasrespect to TAT This approached. on HeLa is a cells was evidenced, their performance on Leishmania parasites challenging human protozoan parasite in reference to peptide membrane interaction. was approached. This Theis a challenging cell membranehuman protozoan of Leishmania parasite by is reinforced in reference a longitudinal to peptide membrane subpellicular layerinteraction. of micro- The cell[39]. tubules membrane of Leishmania This confines all the is reinforcedtraffic membrane by a longitudinal subpellicular to a small membrane arealayer devoidof mi- of crotubules [39]. microtubules, theThis confines flagellar pocket all [40]. the membrane In addition,traffic to a smallwith it is endowed membrane a strongarea devoid proteolytic of microtubules, not armamentarium the only flagellar in thepocket [40].but surface, In also addition, it is endowed in lysosomal enzymes,withespecially a strong proteo- in the lytic armamentarium not only in the surface, but also in lysosomal amastigote form of those species, as L. pifanoi, belonging to the mexicana complex [41,42]. enzymes, especially in thethis To amastigote end, peptides form were of those species, assayed for as L. pifanoi, viability, uptakebelonging to the on and location mexicana complex the protozoan [41,42]. Toparasites. Leishmania this end, peptides were assayed for viability, uptake and location on the pro- tozoan TheLeishmania decrease on parasites. parasite viability was dependent both on the length of the peptide and on Theitsdecrease concentration. on parasiteThe viability toxicity increases with the was dependent length both of the on the peptide length of thefor both peptide forms and on ofits theconcentration. parasite, the dodecamer The toxicity (11) and thewith increases decamer (10) being the length substantially of the peptide formore both toxic than the octamer (9), likely due to the increase of the overall forms of the parasite, the dodecamer (11) and the decamer (10) being substantially more positive charge of the peptide [43]. This inhibitory effect was lower for L. pifanoi amastigotes, toxic than the octamer (9), likely due to the increase of the overall positive charge of the despite of their high content peptide [43].inThis cysteine proteinases inhibitory effectwithwas alower broadfor substrate L. pifanoispecificity, accounting amastigotes, despiteforof5% of their their high total protein content content. in cysteine A similar with proteinases trendawas broadalso observed substrate for their γ, specificity, γ-counterparts, accounting for 5% although withprotein of their total a higher quantitative content. A similar effect [29]. trend was also observed for their γ, γ-counterparts, Because of the nil inclusion of although with a higher quantitative effect [29]. α-amino acids in the peptide composition, the diver- genceBecause among of Leishmania stages is not due the nil inclusion of α-amino acids to differences in the in their proteinase peptide composition, content, but the diver- likely mirrored differences in their membrane architecture. Almost gence among Leishmania stages is not due to differences in their proteinase content, but 40% of the promastig- ote surface likely is covered mirrored with lipophosphoglycan differences in their membrane formed by repetition architecture. Almost of a 40% phosphorylated of the pro- disaccharide mastigote surfaceunit, whereas is covered thewith amastigote hardly expresses lipophosphoglycan formeda few byhundred repetition copies [44]. This of a phosphor- oligosaccharide forms a strong anionic coating that likely ylated disaccharide unit, whereas the amastigote hardly expresses a few hundred acts as an initial anchor point for copies the peptides prior to their internalization. To ascertain the role of length of the peptide in its uptake on L. donovani promastigotes, the set of β,γ-peptides were incubated for 4 h at a fixed concentration of 10 µM. The choice of these conditions relies both on the higher differences in cytotoxicity observed for promastigotes over amastigotes, as well as to the scarce toxicity of the peptides at this point [44]. for Thisthe peptides prior oligosaccharide to their forms internalization. a strong anionic coating that likely acts as an initial anchor Tofor point ascertain the peptidesthe prior role to oftheir length of the peptide in its uptake on L. donovani pro- internalization. mastigotes, the setthe To ascertain of β,γ-peptides role of lengthwere of theincubated peptide in forits4 uptake h at a fixed on L. concentration donovani pro- of 10 mastigotes, μM. The choice the set of β,γ-peptides of these conditions were incubated relies both on forthe 4 h higher at a fixed concentration differences of 10 in cytotoxicity Int. J. Mol. Sci. 2021, 22, 5092 μM. The choice of these conditions relies both on the higher differences observed for promastigotes over amastigotes, as well as to the scarce toxicity of in cytotoxicitythe 7 of 18 pep- observed for promastigotes over amastigotes, as well as to the scarce toxicity tides at this concentration, always less than <20% (Figure 8). According to Figure 9, the of the pep- tides at this concentration, always less than <20% (Figure 8). According to Figure 9, the peptide uptake increased with its length although for all the β,γ-peptides, the uptake was peptide uptake increased with its length although for all the β,γ-peptides, the uptake was consistently concentration,lower thanless always that obtained for CF-TAT at identical concentration, used as a ref- consistently lower than thatthan <20%for obtained (Figure CF-TAT8). According at identicaltoconcentration, Figure 9, the peptide used asuptake a ref- erence for forCPP. increased erence with its length although for all the β,γ-peptides, the uptake was consistently CPP. lower than that obtained for CF-TAT at identical concentration, used as a reference for CPP. Figure 8. Leishmanicidal effect of the peptides. Panel (a) Viability of L. pifanoi amastigotes. Panel (b) L. donovani pro- Figure 8. Leishmanicidal mastigotes. Viability was effect of theafter measured peptides. Panelwith incubation L. pifanoi4amastigotes. (a) Viability of 9–11 Panel (b) L. (20 donovani 6 promastig- Figure 8. Leishmanicidal effect of the peptides. Panel (a)peptides Viability of for h. Leishmania L. pifanoi parasites amastigotes. Panel6 (b)× 10L. cells/mL) donovani pro- otes.incubated were Viability was with measured the afterand peptides, incubation MTT with peptides reduction measured for 4 h. Leishmania 9–11immediately after 4 parasites h (20 × incubation at 10 26 cells/mL) °C at 10 6were (white mastigotes. Viability was measured after incubation with peptides 9–11 for 4 h. Leishmania parasites (20 × 10 cells/mL) incubated column) or with 50 µ the(black M peptides, and final column) MTTpeptide reduction measured immediately concentration. Viability wasafter 4 h incubation represented as the 26 ◦ C at 10of atpercentage (white MTT column) reduc- were incubated with the peptides, and MTT reduction measured immediately after 4 h incubation at 26 °C at 10 (white tion or 50(mean ± SD).column) µM (black Samplesfinal werepeptide made inconcentration. triplicate. Statistical significance Viability respectas was represented tothe untreated control percentage (p ≤ 0.05, of MTT (*); p ≤(mean reduction 0.01, column) or 50 µ M (black column) final peptide concentration. Viability was represented as the percentage of MTT reduc- (**)). ± SD). Samples were made in triplicate. Statistical significance respect to untreated control (p ≤ 0.05, (*); p ≤ 0.01, (**)). tion (mean ± SD). Samples were made in triplicate. Statistical significance respect to untreated control (p ≤ 0.05, (*); p ≤ 0.01, (**)). Figure9.9. Figure UptakeUptake of CF-β,γ-peptides of CF-β,γ-peptides 9–11 by 9–11 by Leishmania Leishmania donovanidonovani promastigotes. promastigotes. Parasites (20Parasites × 106 (20 × 10were6 cells/mL) were incubated for 4 h with the respective peptides at 10 µM. Afterwards, cells/mL) incubated for 4 h with the respective peptides at 10 μM. Afterwards, peptide incor- poration peptide into parasites was incorporation into assessed parasites by wasflow cytometry assessed (λEXC by flow = 488 nm, cytometry λEM == 519 (λEXC 488 nm) and nm, λ EM repre- = 519 nm) sented Figure as 9. the Uptake percentage of with respect CF-β,γ-peptides to CF-TAT 9–11 by ± SD. Error Leishmania bars represent donovani standard promastigotes. and represented as the percentage with respect to CF-TAT ± SD. Error bars represent standard deviation Parasites (20 × 106 (SD) from cells/mL) deviation the were mean value (SD)incubated of three independent for 4 value from the mean h with ofthe experiments respective three independent with peptides each peptide. at 10 μM. experiments Samples withAfterwards, were each peptide.peptide Samplesincor- made were in poration triplicate. into made in Statistical parasites was triplicate. significance assessed Statistical by respect to untreated flow cytometry significance respect control. (λEXC to untreated= 488(pnm, ≤ 0.001, control. λ(pEM≤(***)). =0.001, 519 nm) (***)).and repre- sented as the percentage with respect to CF-TAT ± SD. Error bars represent standard deviation The (SD) fromThe internalization internalization the mean value of ofofthese these three peptides peptidesininexperiments independent Leishmania Leishmaniadonovani donovani with each promastigotes promastigotes was peptide. Samples as- waswere as- sessed madesessed by confocal by confocal in triplicate. microscopy. microscopy. Statistical Thus, promastigotes Thus, promastigotes significance were incubated were incubated respect to untreated with control. (pwith peptides peptides ≤ 0.001, 9–11 (***)).9–11 as aswell well asas TATTATat aatfinal a final concentration concentration of 10ofµM 10 for μM4for h ◦atC.26 h at426 °C. DAPI DAPI wasas was used used as a a nucleic nucleic acid acid dye to dye staintothe stain the nucleus nucleus and theand the kinetoplast kinetoplast (blue fluorescence) (blue fluorescence) (Figure(Figure 10). 10). The internalization of these peptides in Leishmania donovani promastigotes was as- sessed by confocal 2.4. Molecular microscopy. Thus, promastigotes were incubated with peptides 9–11 Modeling as well as TAT at a final concentration of 10 μM for 4 h at 26 °C. DAPI was used as a To ascertain the folding of the peptides in aqueous solution as well as the influence nucleic of theacid dye to stain fluorophore the(CF), moiety nucleus and the molecular kinetoplast dynamics (MD) (blue fluorescence) simulations under an(Figure explicit 10). solvent scenario were carried out on the hybrid β,γ-peptide 8 and its CF-conjugate 11 (MD length of 900 ns and 600 ns, respectively). Both oligomers showed highly disorganized structures along the MD simulations, without any ordered secondary structure motif such as helical or β-sheet-like structures. Considering the backbone carbons of the peptides and a RMSD threshold of 2.0 Å, no cluster accounting for at least 1% of the simulation was found for the peptide 8, whereas the simulation of the CF-conjugate 11 led to twenty clusters Int. J. Mol. Sci. 2021, 22, 5092 8 of 18 containing, at least, 1% of the frames. Consequently, the incorporation of the CF induced a slight structural stabilization of the peptide. An inspection of the representative structures of the two most populated clusters (21% and 8% of the frames) supported the acquisition by the peptide of a globular shape where the peptide wrapped the CF moiety (Figure 11). Int. J. Mol. Sci. 2021, 22, x FOR PEER This REVIEW 8 of 19 trend is observed in a series of MD clusters, but its high dynamics precluded a better identification of any stable interactions between the CF and the peptide. Peptide 11 DAPI Normansky Merge Figure 10. Figure 10. Confocal Confocal microscopy microscopy ofof Leishmania Leishmania donovani donovani promastigotes promastigotesincubated incubatedwith withdodecamer dodecamer11 11 (10 μM final concentration, 4 h,◦26 °C). Afterwards, they were observed without further (10 µM final concentration, 4 h, 26 C). Afterwards, they were observed without further fixation.fixa-CF- tion. CF-peptides (green fluorescence: λEXC = 488 nm/λEM = 519 nm). DAPI (DNA probe, blue fluo- peptides (green fluorescence: λEXC = 488 nm/λEM = 519 nm). DAPI (DNA probe, blue fluorescence: rescence: λEXC = 358 nm/λEM = 461 nm). Magnification bar = 15 μm. λEXC = 358 nm/λEM = 461 nm). Magnification bar = 15 µm. 2.4. Molecular Modeling As mentioned in the introduction, previous studies reported well-defined secondary To ascertain structures for short theoligomers folding ofmade the peptides in aqueous up exclusively withsolution as well as the influence (1S,2S)-2-aminocyclobutane-1- of the fluorophore carboxylic moiety by acid, 5, driven (CF), themolecular formationdynamics (MD) simulations of eight-membered under an explicit hydrogen-bonded rings solvent scenario were carried out on the hybrid β,γ-peptide 8 (Figure 3) [30–33]. Strikingly, no stable hydrogen bonding patterns were found alongand its CF-conjugate 11 (MD length the MDofsimulations, 900 ns and with 600 ns, respectively). a total of 721 and Both oligomers 456 different showed bonds hydrogen highlyobserved disorganized that structures along the MDthroughout appeared/disappeared simulations,the without any ordered trajectories secondary of the peptide 8 andstructure motif such the CF-conjugate 11, respectively. as helical None of structures. or β-sheet-like them stands more than 15% Considering of the time the backbone of simulation, carbons suggesting of the peptides and that eight-membered a RMSD 2.0 Å, rings threshold of stable are either no cluster absent, for accounting or their formation at least 1% of the is quite transitory, simulation was precluding found for the their observation. peptide 8, whereasAnother relevantoffactor the simulation involved in11the the CF-conjugate ledcellular to twentyuptake clus- of CPPs is the cationic character of the peptide, as it endorses a ters containing, at least, 1% of the frames. Consequently, the incorporation of the CF in- high affinity binding into duced thea anionic proteoglycans slight structural exposed stabilization at the of the cell-surface, peptide. mandatory An inspection forrepresentative of the their further internalization structures of the andtwo endosomal escape [45–47]. most populated clusters A proper (21% andorientation of the cationic 8% of the frames) groups supported the facing the solvent acquisition by thewill speed peptide ofup and strengthen a globular shape wherethis interaction. the peptide Inwrapped this regard, themost of the CF moiety time the11). (Figure terminal nitrogen This trend atoms ofinthe is observed guanidinium a series groups are of MD clusters, butexposed its high to the external dynamics pre- aqueous medium according to MD simulations for peptide 8 and cluded a better identification of any stable interactions between the CF and the peptide.its CF-conjugate 11. Apart from visual As mentioned inspection in the of theprevious introduction, representative studies structures reported(Figure 11), a solvent well-defined secondaryac- cessible surface area (SASA) analysis confirmed that the guanidinium structures for short oligomers made up exclusively with (1S,2S)-2-aminocyclobutane-1- groups of oligomers and 11 have 8carboxylic acid,a5,good driven orientation toward the by the formation solvent, with average of eight-membered SASA values hydrogen-bonded of rings 1.145 ± 0.084 2 nm and 1.160 ±no 0.078 2 nmhydrogen , respectively (Table 1). (Figure 3) [30–33]. Strikingly, stable bonding patterns were found along the MD simulations, with a total of 721 and 456 different hydrogen bonds observed that ap- peared/disappeared throughout the trajectories of the peptide 8 and the CF-conjugate 11, respectively. None of them stands more than 15% of the time of simulation, suggesting that eight-membered stable rings are either absent, or their formation is quite transitory, precluding their observation. Another relevant factor involved in the cellular uptake of CPPs is the cationic character of the peptide, as it endorses a high affinity binding into the solvent will speed up and strengthen this interaction. In this regard, most of the time the terminal nitrogen atoms of the guanidinium groups are exposed to the external aqueous medium according to MD simulations for peptide 8 and its CF-conjugate 11. Apart from visual inspection of the representative structures (Figure 11), a solvent Int. J. Mol. Sci. 2021, 22, 5092 accessible surface area (SASA) analysis confirmed that the guanidinium groups of oligo- 9 of 18 mers 8 and 11 have a good orientation toward the solvent, with average SASA values of 1.145 ± 0.084 nm2 and 1.160 ± 0.078 nm2, respectively (Table 1). a) b) c) d) Figure 11. MD simulation of the CF-conjugate 11. Representative snapshots of the two most Figure 11. MD populated simulation clusters for theof the CF-conjugate peptide 11.Ribbon are included. Representative and sticksnapshots of the (a,c), representation two most withpopu- the CF lated clusters for the peptide are included. Ribbon and stick representation (a,c), with the CF motif motif highlighted in purple. Surface representation (b,d), with the CF motif highlighted in purple highlighted in purple. Surface representation (b,d), with the CF motif highlighted in purple and and the terminal nitrogen atoms of the guanidinium groups highlighted in blue. (a,b) Representative the terminal nitrogen atoms of the guanidinium groups highlighted in blue. (a,b) Representative structureof structure ofcluster clusterone. one.(c,d) (c,d)Representative Representativestructure structure of of cluster cluster two. two. Table 1. Table Solventaccessible 1. Solvent accessiblesurface surfacearea area(SASA) (SASA)for for the the guanidinium guanidinium groups groups of of peptide peptide 8 and 8 and CF-CF- conjugate 11 along the MD simulations. conjugate 11 along the MD simulations. Guanidinium Group Guanidinium Peptide 8 CF-Conjugate 11 Peptide 28 1 CF-Conjugate 11 Group SASA (nm ) Percentage SASA (nm2 ) Percentage 1 1 SASA (nm 2) 1.119 Percentage ± 0.240 85.1 ±1 18.3% SASA (nm 1.194 ) ± 20.186 Percentage 90.8 ± 14.1% 1 1 2 1.119 ± 0.2401.105 ± 0.250 84.0 ± 19.0% 1.1941.143 85.1 ± 18.3% ± 0.194 90.8 ± 0.186 86.9± ± 14.8% 14.1% 2 3 1.105 ± 0.2501.083 ± 0.257 82.4 84.0 ± 19.0% ± 19.5% 1.212 ± 1.143 ± 0.194 0.164 92.2 ± 86.9 ± 14.8%12.5% 4 1.147 ± 0.213 87.2 ± 16.2% 1.159 ± 0.201 88.1 ± 15.3% 3 5 1.083 ± 0.257 82.4 ± 19.5% 1.185 ± 0.171 90.1 ± 13.0% 1.2121.154 ± 0.164 ± 0.196 92.2 ± 12.5% 87.8 ± 14.9% 4 6 1.147 ± 0.213 87.2 ± 16.2% 1.232 ± 0.151 93.7 ± 11.5% 1.1591.099± 0.201 ± 0.229 88.1 83.6± ± 15.3% 17.4% 5 Average 1.185 ± 0.171 1.145 ± 0.084 87.1 ± 6.4% 1.1541.160 90.1 ± 13.0% ± 0.078 87.8 ± 0.196 ± 5.9% ± 14.9% 88.2 6 of SASA value 1 Percentage 1.232 ± 0.151 of the 93.7 ± group peptide guanidinium 11.5%over a fully 1.099 ± 0.229 solvated guanidinium83.6 group±(1.315 17.4% nm2 ). Average 1.145 ± 0.084 87.1 ± 6.4% 1.160 ± 0.078 88.2 ± 5.9% Otherwise, 1 Percentage thevalue of SASA influence of theof the spatial peptide arrangement guanidinium group of positively over charged a fully solvated amino acids guanidinium of CPPs on their group (1.315 nm ). 2 translocation profile is still controversial. First, a study on a series of arginine-rich CPPs [23] inferred that large inter-guanidinium distances, up to 15 Å, enhance uptake kinetics.the Otherwise, However, influence a subsequent of the spatialstudy suggested arrangement of that shorter positively distances charged aminofrom 8 to acids 10CPPs of Å were onnecessary to match the their translocation topology profile is stillofcontroversial. anionic groups in cell-surface First, a study onglycans a series[20]. of Indeed, despite arginine-rich CPPsthe[23] conformational flexibility inferred that large distances, upoftoLeishmania of the lipophosphoglycan inter-guanidinium 15 Å , en- donovani, hance as a kinetics. uptake putative However, anchor for aCPPs, a lengthstudy subsequent of 160suggested Å was described for adistances that shorter structure formed by the extended conformation of the 16mer of the phosphorylated from 8 to 10 Å were necessary to match the topology of anionic groups in cell-surface disaccharide, its repetitive forming unit. This accounts for an average separation of 10 Å [48]. In a recent study of ours on γ,γ-CPPs, MD simulations suggested inter-guanidinium distances in the range 8–10 Å both for the most active peptides (Figure 2) or their CF- conjugates [29]. Nevertheless, a similar analysis on 8 and 11 shows average inter-guanidium distances of 14.84 ± 4.46 Å and 16.42 ± 4.17 Å, respectively, not matching the reported as optimal distance among close guanidium groups. These observations offer a possible reason for the poorer activity of the current β,γ-CPPs since they are not prone to form the suitable charge distribution for efficient cell membrane translocation. Int. J. Mol. Sci. 2021, 22, 5092 10 of 18 3. Conclusions A new family of octa-, deca-, and dodecameric hybrid β,γ-peptides and their CF- conjugates were synthesized and evaluated as CPPs for toxicity and cell uptake ability. These peptides were made by a repetitive unit made of a chiral cyclobutane-containing trans-β-amino acid linked to a Nα -functionalized trans-γ-amino-L-proline. Their uptake and cytotoxicity on HeLa cells were not significant. In contrast, toxicity on Leishmania pifanoi amastigotes and especially on Leishmania donovani promastigotes was remarkable at 50 µM, with a toxicity ranking: 12-mer ∼ = 10-mer >8-mer. Peptide internalization by Leishmania was practically nil on L. pifanoi amastigotes and modest on L. donovani promastigotes with a substantial increase with the length of the peptide. By MD simulations, dodecamer 8 and its CF-conjugate 11 are devoid of any conformational bias. These results differ from those reported for their γ,γ-homologues, with a cyclobutane-containing cis-γ-amino acid and a cis- or trans-γ-amino-L-proline as their structural units [29]. This γ,γ-CPP family adopted a well-defined conformation in solution, with modest uptake, and a toxicity on Leishmania parasites higher than the hybrid β, γ-peptides described herein. The SASA analysis of the β,γ-dodecamers (8, and its CF-version, 11) confirmed that the guanidinium groups are exposed to the external aqueous medium for most of the time with an average inter-guanidinium distance of about 15 and 16 Å, respectively. This value did not fall into the interval 8–10 Å described as the optimal distance to match with the anionic groups of cell-surface glycans [20,48]. On the contrary, the γ,γ-counterparts, with a less constrained 1,3-disubstituted cyclobutane moiety, presented more stable conforma- tions with inter-guanidium distances of 8–10 Å for the most active CPPs. Consequently, our results extended the notion of conformational restraint, previously described for α- peptides [49], to lineal CPPs by the inclusion of non-natural amino acids endowed with limited conformational freedom into their sequence. As a conclusion, the use of peptides with tuned conformational restriction, achieved by a rational incorporation of cyclobu- tane β- or γ-amino acids in their sequences, allows the exploration of the topology of guanidium groups and their intramolecular dynamics with a mobility limited by the stiff- ness of the peptide skeleton. Consequently, both stable peptide folding and convenient guanidinium structural patterns are reinforced as CPP key descriptors for an efficient cell-membrane translocation. As such, considering dynamical simulations as a part of future CPPs-engineering protocols appears promising. 4. Materials and Methods The reagents and solvents were purchased from commercial sources and used without further purification. Unless otherwise stated, the reagents were purchased from Sigma Aldrich, Barcelona, Spain. 4.1. Synthesis of the β, γ-Peptides 6–11 and TAT48–57 Peptide β,γ-Peptides were synthesized manually on solid-phase using Aminomethyl-ChemMatrix® resin (f = 0.74 mmol NH2 /g resin). The resin was conditioned with successive washes with DCM (dichloromethane), TFA (trifluoroacetic acid)/DCM (4:5, v/v), DIPEA (N- diisopropylethylamine)/DCM (4:5, v/v), DMF (dimethylformamide) and DCM. The linker was coupled using 3:3:3 Fmoc-Rink amide-Linker/DIC/OxymaPure® (Fmoc = fluorenyl- methoxycarbonyl, DIC = N,N0 -Diisopropylcarbodiimide) in DMF for 1 h. β,γ-Peptides 6-11 backbone was elongated by alternate addition of (2S,4S)-4-Fmoc-amino-1-Alloc-pyrrolidin carboxylic acid (Alloc = Allyloxycarbonyl) and (1S,2S)-2-Fmoc-aminocyclobutane-1-carboxylic acid using DIC/OxymaPure® (3 equiv/3 equiv/3 equiv) as coupling reagents in DMF for 2 h. Fmoc group was released by treatment with piperidine-DMF (2:8, v/v). After each Fmoc elimination step, the peptidyl–resins were washed with DMF and DCM. The couplings were monitored by the Kaiser (ninhydrin) test. Once the peptide backbone syntheses were finished, Alloc groups were eliminated by catalytic reduction using 12/0.1 PhSiH3 /Pd(PPh3 )4 in DCM, and the guanidinium-containing side-chain introduced to the α-amine function of the Amp residues (Amp = 4-aminoproline). Then, the Fmoc Int. J. Mol. Sci. 2021, 22, 5092 11 of 18 group in N-terminal position was eliminated. At this point, the peptidyl-resin was split in two parts. One of these two fractions was conjugated to 5(6)-Carboxyfluorescein using CF/OxymaPure® /PyBOP/DIPEA (4:6:4:6) in DMF (PyBOP = benzotriazol-1- yloxytripyrrolidinophosphonium hexafluorophosphate). Finally, β, γ-peptides 9–11 were cleaved from the peptidyl-resins by treatment with TFA/H2 O/TIS (95:2.5:2.5, v/v/v) (TIS = triisopropylsilane) for 3 h, precipitated in cold diethyl ether, dissolved in CH3 CN/H2 O (1:1, v/v) and lyophilized. The synthesis of TAT48–57 was performed by using standard Fmoc/tert-Bu SPPS methods as previously described [29]. See the Supplementary Materials for more details and Supplementary Tables S1–S3. 4.2. Peptide Purification Crude peptides were purified by semipreparative RP-HPLC-UV-MS using a system composed by a binary gradient Waters 2545, a Waters Alliance 2767 sample manager module and an automatic fraction collector coupled to Waters 2487 dual UV-vis absorbance detector and an electrospray ion source (ESI-MS) Micromass ZQ mass spectrometer detector. The chromatographic separation of the peptides was achieved using a semipreparative column XBridge® Prep BEH C18 (19 × 100 mm, 5 µm) (Waters, Cerdanyola del Vallès, Spain) with dual solvent system formed by A: 0.1% TFA in H2 O and B: 0.1% TFA in CH3 CN at a flow rate of 25 mL/min. λ = 220 nm. Elution gradient was specific for each peptide. 4.3. Peptide Characterization Peptide analysis and determination of the respective retention time (tR ), analytical column XBridge™ BEH 130 C18 (4.6 × 100 mm, 3.5 µM) (Waters, Cerdanyola del Vallès, Spain) was used. Peptide elution was monitored at 220 nm. 4.3.1. Octameric Peptide (6) Purification: RP-HPLC-UV-MS gradient: 5 to 40% B in 10 min; 40 to 60% B in 1 min, and to 100% B in 2 min. Characterization: RP-HPLC. Purity >95%, tR 3.4 min. m/z (ESI): Ms Calcd for C64 H107 N25 O12 [M + 3H/3]+ : 474.6; Experimental: 473.2. Ms Calcd para C64 H107 N25 O12 [(M + 4H)/4]+ : 355.6; Experimental: 355.7. Ms Calcd for C64 H107 N25 O12 [(M + 5H)/5]+ : 284.7; Experimental: 284.7. MALDI-TOF: Ms Calcd for C64 H107 N25 O12 [(M + H)]+ : 1419.72; Experimental: 1419.02. Ms Calcd para C64 H107 N25 O12 [M + Na]+ : 1457.82; Experimental: 1457.00. 4.3.2. Decameric Peptide (7) Purification: RP-HPLC-UV-MS gradient: 5 to 40% B in 10 min; 40 to 60% B in 1 min, and to 100% B in 2 min. Characterization: RP-HPLC. Purity >98%, tR 3.4 min. m/z (ESI): Ms Calcd for C80 H133 N31 O15 [(M + 3H)/3]+ : 590.7; Experimental: 590.7. Ms Calcd for C80 H133 N31 O15 [(M + 4H)/4]+ : 445.2; Experimental: 445.3. Ms Calcd for C80 H133 N31 O15 [(M + 5H)/5]+ : 354.8; Experimental: 354.7. MALDI-TOF: Ms Calcd for C80 H133 N31 O15 [M + H]+ : 1769.06; Experimental: 1769.22. 4.3.3. Dodecameric Peptide (8) Purification: RP-HPLC-UV-MS gradient: 10 to 60% B in 11 min, and to 100% B in 2 min. Characterization: RP-HPLC. Purity >99%, tR 3.5 min. m/z (ESI): Ms Calcd for C96 H159 N37 O18 [(M + 4H)/4]+ : 530.8; Experimental: 530.9. Ms Calcd for C96 H159 N37 O18 [(M + 5H)/5] + : 424.9; Experimental: 424.8. MALDI-TOF: Ms calcd for C96 H159 N37 O18 [M + H]+ : 2119.27; Experi- mental: 2119.47. Ms Calcd for C96 H159 N37 O18 [M + Na]+ : 2141.26; Experimental: 2141.40. 4.3.4. Octameric CF-Peptide (9) Purification: RP-HPLC-UV-MS gradient: 5 to 40% B in 10 min; 40 to 60% B in 1 min, and to 100% B in 2 min. Characterization: RP-HPLC. Purity >99%, tR 5.9 min. m/z (ESI): Ms Calcd for C85 H117 N25 O18 [(M + 2H)/2]+ : 889.5; Experimental: 889.3. Ms Calcd for C85 H117 N25 O18 [(M + 3H)/3]+ : 593.3; Experimental: 593.7. Ms Calcd para C85 H117 N25 O18 [(M + 4H)/4]+ : Int. J. Mol. Sci. 2021, 22, 5092 12 of 18 445.2; Experimental: 445.1. Ms Calcd for C85 H117 N25 O18 [(M + 5H)/5]+ : 356.4; Experimental: 356.3. MALDI-TOF: Ms calcd for C85 H117 N25 O18 [(M + 3H)]+ : 1778.03; Experimental: 1778.20. Ms Calcd for C85 H117 N25 O18 [M + Na]+ : 1800.02; Experimental: 1799.22. 4.3.5. Decameric CF-Peptide (10) Purification: RP-HPLC-UV-MS gradient: 5 to 50% B in 10 min; 50 to 60% B in 1 min, and to 100% B in 2 min. Characterization: RP-HPLC. Purity >99%, tR 5.5 min. m/z (ESI): Ms Calcd for C101 H143 N31 O21 [(M + 3H)/3]+ : 710.1; Experimental: 710.4. Ms Calcd for C101 H143 N31 O21 [(M + 4H)/4]+ : 532.8; Experimental: 532.8. Ms Calcd for C101 H143 N31 O21 [(M + 5H)/5]+ : 426.4; Experimental: 426.4. MALDI-TOF: Ms Calcd for C101 H143 N31 O21 [M + H]+ : 2127.11; Experimental: 2127.32. 4.3.6. Dodecameric CF-Peptide (11) Purification: RP-HPLC-UV-MS gradient: 10 to 60% B in 11 min, and to 100% B in 2 min. Characterization: RP-HPLC. Purity >99%, tR 4.8 min. m/z (ESI): Ms Calcd for C117 H169 N37 O24 [(M + 3H)/3]+ : 826.9; Experimental: 826.5. Ms Calcd for C117 H169 N37 O24 [(M + 4H)/4]+ : 620.4; Experimental: 620.3. Ms Calcd for C117 H169 N37 O24 [(M + 5H)/5]+ : 496.5; Experimental: 496.5. MALDI-TOF: Ms Calcd for C117 H169 N37 O24 [M + H]+ : 2477.31; Experimental: 2477.46. 4.4. Cellular Viability, Internalization, and Localization Experiments with HeLa Cells The HeLa cell line, derived from a human cervical cancer, was used to perform the biological assays. CPP dissolved in non-supplemented Minimum Essential Medium (MEM) (Gibco, Thermo Fisher Scientific, Cornellà de Llobregat, Spain) at their final concentration were sterilized by filtration through a Whatman® Puradisc 0.2 µm polycarbonate filter. 4.4.1. HeLa Cells Culture The cells were cultured in 25 cm2 flasks in MEM culture media supplemented with 10% of heat inactivated fetal bovine serum (FBS, Gibco) plus 2 mM L-glutamine (Biowest, Labclinics, Barcelona, Spain) at 37 ◦ C, saturated humidity and 5% of CO2 (standard conditions). 4.4.2. Cellular Viability The MTT assay was used to assess the cytotoxicity of the peptides. This method is based on the ability of living cells to reduce the MTT to formazan salts by mitochon- drial reductases. HeLa cells were seeded into 24 microwell plates at 6 × 104 cells/mL (30,000 cells/well). After 24 h of incubation, the medium was replaced with fresh medium with the respective peptide concentration and incubated for 24 h. Then, the cells were washed three times with Hanks buffered saline solution (HBSS) (Biowest, Labclinics, Barcelona, Spain), and 500 µL of 0.1 mg/mL MTT in HBSS were added to the cell sus- pension and incubated for 3 h at 37 ◦ C in darkness. The cell layer was dried in darkness, and the resulting formazan solubilized in pure DMSO. The absorbance was measured at 540 nm in an X3 Multilabel Plate Reader coupled to Perkin Elmer 2030 Manager control software. At least three independent experiments with four different replicates of each peptide and concentration were performed. Controls, non-treated cells, or cells incubated with CF were included at identical concentrations. The absorbance of non-treated cells was taken as 100% cellular viability. 4.4.3. Peptide Internalization For flow cytometry experiments, HeLa cells were seeded into 35 mm culture dishes (2 × 105 cells/dish). After 24 h incubation under standard conditions, culture medium was removed, and cells were incubated for 2 h with the corresponding peptides at 10 and 25 µM. Next, the culture medium was removed, the cells were washed twice with HBSS and then trypsinized with 0.5 mL of 0.25% trypsin-EDTA (Gibco, Thermo Fisher Scientific, Cornellà de Llobregat, Spain). After 5 min incubation at 37 ◦ C, 2 mL of MEM + 10% FCS was Int. J. Mol. Sci. 2021, 22, 5092 13 of 18 added to the cells to stop trypsinization and the mixture was centrifuged (5 min, 300× g). The cells were then additionally washed with 2 mL of HBSS under the same conditions. Finally, the cell pellet was resuspended in 200 µL of PBS at pH = 6.0 to detach any peptide adhering to the plasma membrane. To exclude dead cells from gating, 5 µg/mL propidium iodide (PI, Sigma-Aldrich, Barcelona, Spain) was added to the cells immediately before the flow cytometric analysis, carried out in a BD FACSCanto cytometer (Bio-Rad, Alcobendas, Spain) coupled to FACSDiva v.7.0 software using 488 nm and 635 nm lasers to excite the peptides and PI, respectively. A total of 10,000 single cells were analyzed per sample, and at least three independent experiments were performed with each peptide and concentration. Untreated cells (autoflu- orescence control) and cell cultures incubated with TAT as positive reference CPP, and CF as negative reference, were included in each experiment. The fluorescence intensity of cells treated with CF was taken as the arbitrary unit for normalization. For confocal microscopy, HeLa cells were seeded into glass bottom culture dishes (MatTek, Bratislava, Slovakia) at a density of 2 × 105 cell/dish. After 24 h of incubation, the culture medium was removed, and the cells were incubated for 2 h in the presence of peptides at 25 µM. Then, cells were rinsed three times with PBS, and nuclei and plasma membrane were counterstained with 1 µL/mL of Hoechst 33,342 (10 mg/mL, Thermo Fisher, Scientific, Cornellà de Llobregat, Spain) and 1 µL/mL CellMask™ deep red plasma membrane stain (5 mg/mL, Thermo Fisher Scientific, Cornellà de Llobregat, Spain), respectively. Finally, the cells were washed with PBS prior to be resuspended in PBS pH = 6.0. The experiments were performed using an Olympus Fluoview FV1000 confocal laser scanning microscope (Olympus Iberia, Hospitalet de Llobregat, Spain) equipped with Olympus Fluoview as control software. The excitation wavelengths used were 405, 488, and 658 nm to visualize the nuclei, the peptides and the plasma membrane, respectively; the wavelength of emission was 460, 510 and 690 nm, respectively. A 3D reconstruction was generated to obtain orthogonal projections using the ImageJ/fiji software. 4.5. Cellular Viability, Internalization, and Localization Experiments with Leishmania Parasites The experiments were carried out with Leishmania donovani promastigotes (MHOM/SI/ 00/1S-2D strain) and Leishmania pifanoi amastigotes (MHOM/VE/60/Ltrod strain), respec- tively. Promastigotes were grown at 26 ◦ C in RPMI medium supplemented with 10% of FCS, 2 mM L-glutamine, 20 U/mL unicilin 20 plus 48 µg/mL gentamicin. 4.5.1. Cellular Viability The viability assays were carried out using the reduction of MTT as described. The par- asites were aliquoted into 96-microwell plates at a final concentration of 20 × 106 cells/mL in HBSS supplemented with 10 mM D-glucose (Glc). The peptides were incubated for 4 h at the corresponding peptide concentration at 26 ◦ C or 32 ◦ C for promastigotes and axenic amastigotes, respectively. Afterwards, 0.5 mg/mL MTT in HBSS + 10 mM Glc was added and the cells were incubated for two additional hours. The resulting formazan was solubilized with DMSO (1% final concentration) and read at 595 nm in a Bio-Rad 640 microplate reader. 4.5.2. Peptide Uptake for Leishmania donovani Promastigotes The parasites were resuspended in HBSS + Glc and dispensed in 24-microwell plates (2 mL/well) at a final concentration of 20 × 106 cells/mL. After incubation with the peptides for 4 h at 26 ◦ C, the parasites were washed twice with 2 mL of HBSS + Glc plus 1% fatty-acid free bovine seroalbumin in order to remove the non-internalized peptides and resuspended in the same medium at 1 × 106 cells/mL. Propidium iodide (PI) at a final concentration of 5 µg/mL was added immediately to the flow cytometric analysis to gate the viable cells exclusively. Flow cytometry was carried out in a FC500 flow cytometer, using λEXC = 488 nm and λEM = 525 nm for fluoresceinated peptides and λEXC = 488 nm. Int. J. Mol. Sci. 2021, 22, 5092 14 of 18 4.5.3. Intracellular Localization of the Internalized Peptides in Leishmania donovani Promastigotes The parasites (2 × 106 cells/mL in HBSS + Glc, 200 µL) were incubated with the corresponding peptides (2 h, 26 ◦ C). Afterwards, they were collected from the well and washed twice with 2 mL of HBSS + Glc plus 1% bovine seroalbumin fatty-acid free. Prior to carrying out confocal microscopy, the parasites were incubated with 10 µL/mL of DAPI (40 ,6-diamidine-20 -phenylindole dihydrochloride) for 10 min at 26 ◦ C and washed twice with 2 mL of HBSS + Glc. Living parasites were observed using a Leica SP2 confocal microscope (Leica Microsistemas, S.A., Barcelona, Spain) at λEXC = 358 nm/λEM = 461 nm. 4.5.4. Statistical Treatment Statistical significance was inferred with t-test, using the statistics platform of the SigmaPlot v.11.0 software: ((p ≤ 0.05, (*); p ≤ 0.01, (**); p ≤ 0.001, (***)). 4.6. Computational Details Geometries without steric clashes of the hybrid β, γ-peptides 8 and 11 were obtained through RDKit ETKDGv3 conformer generation [50], taking the conformer with lowest energy as the initial structure for the MD simulations. Atomic charges were computed with the restrained electrostatic potential (RESP) protocol [51]. The atom types and force field parameters were assigned through antechamber and parmchk2 tools of the AmberTools20 package [52]. The peptides were solvated with a cubic box of TIP3P water molecules and the positive charges of the guanidinium groups were neutralized with six chloride ions in each case, employing the ions94 library. The GAFF force field [53] was used for all atoms of the peptides. MD simulations were carried out under periodic boundary conditions with the OpenMM engine [54] using the OMMProtocol application [55]. The convergence of the trajectories was assessed by RMSD, all-to-all RMSD, PCA and cluster counting analyses [56], taking the exploration of the conformational space defined by the twelve backbone carbons of the peptides (see Supplementary Materials, Table S4 and Figures S1 and S2 for further details). In particular, the simulation of the β, γ- peptide 8 was extended up to 900 ns, whereas the β, γ-peptide 11 simulation was of 600 ns length. UCSF Chimera [57] was used to visually analyze the trajectories. Pytraj from the AmberTools20 package [53] was used for the hydrogen bond detection and guanidinium distance calculation. Quality threshold clustering [58], with a threshold of 2.0 Å and a selection of the alpha carbons, was used to generate the clusters and the representative structures. The SASA values of the guanidinium groups (carbon, two terminal nitrogen atoms, and their corresponding hydrogen atoms) were calculated with the MDTraj [59] implementation of the Shrake and Rupley’s algorithm. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms22105092/s1, Synthesis of the monomers for solid phase peptide synthesis (SPPS); NMR spectra of the new monomers; SPPS procedures; HPLC chromatograms and MS spectra of the purified peptides 6–11; convergence of Molecular Dynamics simulations; Table S1. General protocol for the synthesis of the peptides. Table S2. General protocol for the derivatization of the α-amine function using the solid phase synthesis. Table S3. General protocol for the incorporation of the 5(6)-carboxyfluorescein. Table S4: Specifications of the system for the MD simulations; Figure S1: Convergence studies for the MD simulation of peptide 8. (a) RMSD against the first frame of the trajectory. (b) Principal component analysis. Plot of the first principal component against the second principal component along the MD. (c) Cluster counting along the MD with a cluster cutoff of 5.0 Å. (d) All-to-all frames RMSD along the MD; Figure S2: Convergence studies for the MD simulation of CF-conjugate 11. (a) RMSD against the first frame of the trajectory. (b) Principal component analysis. Plot of the first principal component against the second principal component along the MD. (c) Cluster counting along the MD with a cluster cutoff of 5.0 Å. (d) All-to-all frames RMSD along the MD. Author Contributions: Conceptualization, O.I., J.-D.M., M.R. and R.M.O.; Funding acquisition, O.I., C.N., L.R., J.-D.M., M.R. and R.M.O.; Investigation, J.O., J.-E.S.-A., X.P., D.C., M.Á.A., N.G., L.R. and J.-D.M.; Methodology, C.N., L.R., J.-D.M. and M.R.; Supervision, O.I., J.-D.M., M.R. and R.M.O.; Int. J. Mol. Sci. 2021, 22, 5092 15 of 18 Writing—Original draft, R.M.O.; Writing—Review & editing, O.I., J.-E.S.-A., C.N., L.R., J.-D.M., M.R. and R.M.O. All authors have given approval to the final version of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Spanish Ministry of Economy, Industry and Competitivity (Grants CTQ2016-77978-R, MAT2017-86357-C3-3-R, SAF2014-60138-R, and RTI2018-093831-B-I00), Generalitat de Catalunya (2017-SGR-503 and 2017-SGR-1439), Subdirección General de Redes y Centros de Investigación Cooperativa-FEDER (RICET RD16/0027/0010) and CSIC (PIE 201620E038) and CIBER BBN (CB/06/01/0074). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. The data are not publicly available due to their use in further studies. Acknowledgments: The authors would like to thank the staff at the Servei de Microscòpia de la Universitat Autònoma de Barcelona and the Peptide Synthesis Unit of the NANBIOSIS ICTS (U3, CIBER BBN-IQAC-CSIC) for technical assistance. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Abbreviations ACN Acetonitrile Alloc Allyloxycarbonyl Amp 4-Aminoproline CBAA Cyclobutane Amino Acid CF 5(6)-Carboxyfluorescein CPP Cell Penetrating Peptide DAPI 40 ,6-Diamidino-2-phenylindole DCM Dichloromethane DDS Drug Delivery Systems DFT Density Functional Theory DIC N,N0 -Diisopropylcarbodiimide DIPEA N-Diisopropylethylamine DMF Dimethylformamide DMSO Dimethyl Sulfoxide EDTA Ethylenediaminetetraacetic Acid ESI Electrospray Ionization FBS Fetal Bovine Serum Fmoc Fluorenylmethyloxycarbonyl Glc Glucose HBSS Hanks Buffered Saline Solution HPLC High-Performance Liquid Chromatography LPG Lypophosphoglycan MALDI Matrix Assisted Laser Desorption Ionization MS Mass Spectroscopy MD Molecular Dynamics MEM Minimum Essential Medium MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide NMR Nuclear Magnetic Resonance PBS Phosphate-Buffered Saline PCA Principal Component Analysis PES Potential Energy Surface PI Propidium Iodide Int. J. Mol. Sci. 2021, 22, 5092 16 of 18 PyBOP (7-Azabenzotriazol-1-yloxy)-tripyrrolidinophosphonium Hexafluorophosphate RESP Restrained Electrostatic Potential RMSD Root-Mean-Square Deviation RMSF Root-Mean-Square Fluctuation RP Reverse Phase SASA Solvent Accessible Surface Area SD Standard Deviation SM Supplementary Materials SPPS Solid Phase Peptide Synthesis TIS Triisopropylsilane TOF Time of Flight UV Ultraviolet Spectroscopy References 1. Lindgren, M.; Hällbrink, M.; Prochiantz, A.; Langel, Ü. Cell-penetrating Peptides. Trends Pharmacol. Sci. 2000, 21, 99–103. [CrossRef] 2. Langel, Ü. Cell-Penetrating Peptides in Processes and Applications; CRC Press Pharmacology and Toxicology Series; CRC Press: Boca Raton, FL, USA, 2002. [CrossRef] 3. Lundberg, P.; Langel, Ü. A brief introduction to cell-penetrating peptides. J. Mol. Recognit. 2003, 16, 227–233. [CrossRef] 4. Kim, G.C.; Cheon, D.H.; Lee, Y. Challenge to overcome current limitations of cell-penetrating peptides. Biochim. Biophys. 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