TYPE Original Research PUBLISHED 15 November 2022 DOI 10.3389/fphar.2022.1060827 Cell-penetrating OPEN ACCESS peptide-conjugated copper EDITED BY Yong Zhang, Jilin University, China complexes for redox-mediated REVIEWED BY Jing Sun, anticancer therapy Geneleap Biotech, Luye Pharm, China Ji Li, University of Michigan, United States Quim Peña 1,2,3†, Sergi Rodríguez-Calado 4†, A. Jalila Simaan 2, *CORRESPONDENCE Mercè Capdevila 1, Pau Bayón 1, Oscar Palacios 1, Julia Lorenzo 4* Julia Lorenzo, and Olga Iranzo 2* julia.lorenzo@uab.cat Olga Iranzo, 1 Departament de Química, Facultat de Ciències, Universitat Autònoma de Barcelona, Barcelona, Spain, olga.iranzo@univ-amu.fr 2 Aix Marseille University, CNRS, Centrale Marseille, ISm2, Marseille, France, 3Department of † Nanomedicine and Theranostics, Institute for Experimental Molecular Imaging, Faculty of Medicine, These authors have contributed equally RWTH Aachen University Clinic, Aachen, Germany, 4Department Bioquímica i Biologia Molecular, to this work Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, Barcelona, Spain SPECIALTY SECTION This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Metal-based chemotherapeutics like cisplatin are widely employed in cancer Frontiers in Pharmacology treatment. In the last years, the design of redox-active (transition) metal RECEIVED 03 October 2022 complexes, such as of copper (Cu), has attracted high interest as alternatives to ACCEPTED 28 October 2022 PUBLISHED 15 November 2022 overcome platinum-induced side-effects. However, several challenges are still faced, including optimal aqueous solubility and efficient intracellular delivery, and CITATION Peña Q, Rodríguez-Calado S, strategies like the use of cell-penetrating peptides have been encouraging. In this Simaan AJ, Capdevila M, Bayón P, context, we previously designed a Cu(II) scaffold that exhibited significant reactive Palacios O, Lorenzo J and Iranzo O (2022), Cell-penetrating peptide- oxygen species (ROS)-mediated cytotoxicity. Herein, we build upon the promising conjugated copper complexes for Cu(II) redox-active metallic core and aim to potentiate its anticancer activity by redox-mediated anticancer therapy. rationally tailoring it with solubility- and uptake-enhancing functionalizations that do Front. Pharmacol. 13:1060827. doi: 10.3389/fphar.2022.1060827 not alter the ROS-generating Cu(II) center. To this end, sulfonate, arginine and COPYRIGHT arginine-rich cell-penetrating peptide (CPP) derivatives have been prepared and © 2022 Peña, Rodríguez-Calado, characterized, and all the resulting complexes preserved the parent Cu(II) Simaan, Capdevila, Bayón, Palacios, coordination core, thereby maintaining its reported redox capabilities. Lorenzo and Iranzo. This is an open- access article distributed under the Comparative in vitro assays in several cancer cell lines reveal that while specific terms of the Creative Commons solubility-targeting derivatizations (i.e., sulfonate or arginine) did not translate into an Attribution License (CC BY). The use, distribution or reproduction in other improved cytotoxicity, increased intracellular copper delivery via CPP-conjugation forums is permitted, provided the promoted an enhanced anticancer activity, already detectable at short treatment original author(s) and the copyright times. Additionally, immunofluorescence assays show that the Cu(II) peptide- owner(s) are credited and that the original publication in this journal is conjugate distributed throughout the cytosol without lysosomal colocalization, cited, in accordance with accepted suggesting potential avoidance of endosomal entrapment. Overall, the academic practice. No use, distribution systematic exploration of the tailored modifications enables us to provide further or reproduction is permitted which does not comply with these terms. understanding on structure-activity relationships of redox-active metal-based (Cu(II)) cytotoxic complexes, which contributes to rationalize and improve the design of more efficient redox-mediated metal-based anticancer therapy. KEYWORDS cell-penetrating peptide, copper, metal complex, intracellular delivery, cancer, redox- active, metallodrug Frontiers in Pharmacology 01 frontiersin.org Peña et al. 10.3389/fphar.2022.1060827 GRAPHICAL ABSTRACT Modulation of solubility and intracellular delivery of redox-active copper complexes via cell-penetrating peptide-conjugation enhances cytotoxicity. Introduction generate radical species, occur, to a considerable extent, at the intracellular level. Additionally, metal complexes do often face Metal-based chemotherapeutics have shown high clinical relevance solubility issues that contribute to limit its real applicability in cancer therapy due to the possibility of tackling diverse (sub)cellular (Savjani et al., 2012). Hence, and given that many targets and acting via multiple mechanisms of action thanks to the chemotherapeutics are administered intravenously, solubility and unique metal-ligand interplay (Anthony et al., 2020). Platinum (Pt) intracellular delivery appear as two important features in the complexes such as cisplatin, carboplatin and oxaliplatin have already design of potential redox-active (Cu) chemotherapeutics. become first-line treatments for several types of cancer (Rottenberg To help overcome these challenges, the use of delivery systems or et al., 2021). Despite their success, the high reactivity, lack of specificity functional moieties that increase solubility and enhance intracellular and non-physiological nature of Pt often lead to undesired biological delivery, such as viral-based vectors, vesicles, nanoparticles, or cell- interactions and side-effects (de Luca et al., 2019; Ohmichi et al., 2005; penetrating peptides (CPPs) can be used (Allen and Cullis, 2004; Oun et al., 2018; Wheate et al., 2010; Zhou et al., 2020). To overcome Dhar et al., 2009; Nakase et al., 2012; Stewart et al., 2018; Tran et al., some of these obstacles, the use of metal complexes bearing 2017; Vargason et al., 2021; Zhou & Kopeček, 2013) Concretely, the physiological metals such as copper (Cu) have arisen as promising latter is one of the most common and promising strategies to deliver alternatives. In addition to the enhanced biocompatibility of Cu, its therapeutics inside cancer cells. CPPs comprise a class of short complexes can induce toxicity via diverse mechanisms beyond the peptides (<30 amino acids) that have the ability to cross cellular common DNA-binding mode of action of Pt(II) complexes, such as membranes (Schmidt et al., 2010; Guidotti et al., 2017). They have DNA cleavage, topoisomerase inhibition and oxidative (mitochondrial) acquired considerable attention due to their high transduction disruption (Santini et al., 2014). In particular, the different biologically efficiency and low associated cytotoxicity (Guo et al., 2016; attainable oxidation states of Cu enable the preparation of what is Guidotti et al., 2017; Borrelli et al., 2018). More than 80% of the broadly known as catalytic metallodrugs, i.e., metal complexes that known CPPs are cationic and contain more than five positively mediate catalytic reactions in biological environments, including the charged amino acids, being arginine (Arg)-rich CPPs the most widely generation of reactive oxygen species (ROS). studied class by far (Schmidt et al., 2010; Milletti, 2012; Raucher and ROS-based chemotherapy has awakened extensive interest due Ryu, 2015). Multiple mechanisms, highly dependent on the to the possibility of promoting selectivity towards cancer cells, which physicochemical nature and number of amino acid residues, have is based on the higher basal ROS levels in cancer than in healthy cells been reported for CPPs, including translocation, diffusion and (Liou and Storz, 2010), and of bypassing potential drug resistance endocytosis (Brock, 2014). The positively charged guanidinium mechanisms (Cui et al., 2018). Additionally the generally more groups in the arginine residues have high affinity for the reducing environment of cancer cells (Petrova et al., 2018) can externally-faced negatively charged fatty acids of the cell favor the reduction of Cu(II), thereby triggering the catalytic cycle. membrane, mediating the peptide insertion into the cytosol Several reported Cu (and other metal) complexes have already shown (Rothbard et al., 2004; Herce et al., 2014). One of the key issues promising therapeutic performance through ROS-based mechanisms in efficient intracellular delivery of CPPs is the subsequent endosomal of action (Li et al., 2020; Liu et al., 2019; Peña et al., 2019; Simunkova escape. Most of the reported CPPs are able to promote et al., 2019; Sîrbu et al., 2017). Nonetheless, most Cu- and other internalization, but the mechanisms for which they can escape metal-related biological pathways, including catalytic processes to from endosomes are still not completely understood. Among Frontiers in Pharmacology 02 frontiersin.org Peña et al. 10.3389/fphar.2022.1060827 FIGURE 1 (A) Schematic representation of the work rationale. Assessing the impact of the different functionalizations on the uptake and anticancer activity of Cu(II) complexes. (B) Structure of the Cu(II) complex C1 (Peña et al., 2021) and the different H2L1 functionalized ligands (H2L2-H2L5), where R and G in the peptides structure correspond to arginine (Arg) and glycine amino acids, respectively. The name of the Cu(II) complex corresponding to each ligand is specified in parentheses. them, strategies encompassing the use of cationic structures (such as with the main goal of increasing solubility in aqueous media, Arg-rich based) and/or amphiphilic structures that can disrupt namely the addition of a sulfonate group (ligand H2L2 and endosomal membranes because of the high electrostatic charge complex C2), and of an arginine (Arg) residue (ligand and partial hydrophobicity have been reported (Brock et al., 2018; H2L3 and complex C3) (Figure 1B). Both groups are Najjar et al., 2017). biologically relevant and introduce a charge of opposite sign Taking all into account, we here aim to (1) explore feasible, at physiological pH, i.e., negative for sulfonate group and positive rational and biologically relevant functionalization strategies that for Arg. Taking into account that the number of Arg residues tackle the solubility and intracellular delivery issues that many affects the internalization ability of CPPs (being those with at (redox-active) Cu complexes face while, at the same time, (2) least six Arg residues more efficient (Wender et al., 2000; systematically assessing (and eventually rationalizing) the impact of Tünnemann et al., 2008; Raucher and Ryu, 2015; Guidotti these specific solubility- and uptake-promoting modifications in the et al., 2017)), and in order to tackle potential cellular uptake activity of the complex against cancer cells. To evaluate this, we built limitations as well, two additional modifications were also upon a Cu(II) N,N,O-chelating salphen-like complex (C1), recently evaluated: a nine Arg residues peptide (R9, ligand H2L4 and reported by our group (Peña et al., 2021). Despite exhibiting some complex C4) and its analogous sequence with four glycine (G) solubility issues, C1 was observed to display a remarkable redox- residues (G4-R9, ligand H2L5 and complex C5) (Figure 1B). R9 mediated cytotoxicity, and a putative discrimination between cancer was selected for its well-known good cell-penetrating capabilities and healthy cells, through the generation of ROS. To this end, we (Wender et al., 2000; Guidotti et al., 2017), and glycine residues established simple, modular and versatile functionalization were introduced to test the effect of having a linker between the methodologies to prepare several tailored derivatives of the parent CPP and the metallic core. Additionally, the cationic nature of ligand of the complex C1 (i.e., H2L1), with ideally no or minimal the CPPs, in combination with the relatively more hydrophobic alteration of the metal-ligand interaction, known to play a key role in salphen-based metallic core, may help address possible endo-/ determining the final (re)activity. lysosomal entrapment issues (Brock et al., 2018). Whilst using The rationale of our work is schematically depicted in here C1 as our model parent scaffold, we expect that the obtained Figure 1A and the specific derivatizations shown in Figure 1B. data will contribute to further rationalize the role of the different First, two different functionalization strategies were explored functionalities in tuning the intracellular delivery and Frontiers in Pharmacology 03 frontiersin.org Peña et al. 10.3389/fphar.2022.1060827 corresponding activity of analog redox-active Cu(II) complexes, High-performance liquid chromatography overall providing a starting point for the design of future (HPLC) improved ROS-inducing (Cu) metallodrugs. Reversed-phase HPLC analyses were carried out on an Agilent Technologies 1,200 Series with a ultraviolet-visible (UV-vis) detector. Materials and methods A C12 Jupiter Proteo (90Å, 4 μm, 250 × 4.6 mm) and a C12 Jupiter Proteo Axia (90Å, 4 μm, 250 × 21.2 mm) columns were used for the Chemicals and reagents analytical and preparative scales, respectively. A two-solvent gradient was used (Solvent A: H2O/TFA (99.9/0.1%); and Solvent B (ACN/ Reagents like copper(II) chloride, copper(II) acetate, sodium H2O/TFA (90/9.9/0.1%)). Absorbance at 220, 260 and 310 nm were carbonate, sodium sulfate, hydrochloric acid, ammonium chloride, used to monitor the different compounds. Details of the gradients o-phenylenediamine, 2-amino-2-(hydroxymethyl)propane-1,3-diol used are given in the corresponding synthetic procedures. (TRIS), N,N-diisopropylethylamine (DIEA), triisopropylsilane (TIS), N-methyl-2-pyrrolidone (NMP) and piperidine were obtained from Sigma-Aldrich® and Thermofisher®. Solvents such Nuclear magnetic resonance (NMR) as acetonitrile (ACN), methanol (MeOH), ethanol (EtOH), diethyl spectroscopy ether (Et2O), chloroform (CHCl3), dimethyl sulfoxide (DMSO), N,N- dimethylformamide (DMF), ethyl acetate (EtOAc), dichloromethane NMR experiments were recorded on BRUKER DPX-250, (DCM), acetic anhydride and hexane were used at synthesis grade 300, 360, 400 and 500 MHz instruments at the Servei de purity and directly from commercial sources (Scharlab®, Panreac® Ressonància Magnètica Nuclear (UAB) and Spectropole and VWR®). Trifluoroacetic acid (TFA) was purchased from Acros facilities (AMU). Deuterated solvents were directly purchased Organics®. The N-fluorenylmethoxycarbonyl (Fmoc)-protected from commercial suppliers. All spectra have been registered at amino acids (Fmoc-Arg (Pbf)-OH and Fmoc-Gly-OH), 2- 298 K otherwise noticed. The abbreviations used to describe (1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium signal multiplicities are: s (singlet), d (doublet), dd (double hexafluorophosphate (HBTU) and the Rink amide MBHA doublet), t (triplet), bs (broad signal) and m (multiplet). All (100–200 mesh) resin were obtained from Novabiochem®. 13 C NMR acquired spectra are proton decoupled. Synthetic protocols Mass spectrometry (MS) Ligand H2L1 and its corresponding complex C1 were re- Routine electrospray ionization (ESI)-MS measurements were synthesized and characterized as previously described (Peña recorded at the Spectropole facility (AMU) on a SYNAPT G2 HDMS et al., 2021). The synthesis of ligands H2L2-H2L5 and the (Waters) instrument with an ionization source at atmospheric corresponding copper(II) complexes (C2-C5) is described in pressure (API) pneumatically assisted and with a time-of-flight the Supplementary Material S1. analyzer (TOF). Ionization conditions: electrospray voltage of Peptides (R9 and G4-R9) were synthesized using a microwave 2.8 kV, capillary voltage of 20 V, dry gas flow at 100 L/h. High assisted Biotage® Initiator + Alstra synthesizer, following standard resolution (HR)-MS measurements were recorded after diluting the Fmoc solid-phase peptide synthesis (SPPS) protocols (Chan & corresponding solid complexes in a MicroTOF-Q (Brucker Daltonics White, 2000). They were synthesized on a Rink amide MBHA GmbH, Bremen, Germany) instrument equipped with an electrospray resin (100–200 mesh) in a 0.25 mmol scale (0.59 mmol/g). The ionization source (ESI) in positive mode at the Servei d’Anàlisi amino acids (4 eq) were assembled using HBTU (3.9 eq) as coupling Química (UAB). The nebulizer pressure was 1.5 Bar, the agent, DIEA (8 eq in N-methyl-2-pyrrolidone (NMP)) as a base and desolvation temperature was 180°C, dry gas at 6 L min−1, the DMF as a solvent. Fmoc deprotection was carried out at room capillary counter-electrode voltage was 5 kV and the quadrupole temperature with 20% piperidine in DMF for about 20 min. ion energy, 5.0 eV. Couplings were carried out at 75°C for Fmoc-Gly-OH (5 min). For Fmoc-Arg (Pbf)-OH residues, double couplings were carried out at 50°C (2 × 6.5 min). Cleavage and simultaneous removal of the Electron paramagnetic resonance (EPR) protecting groups were done manually with a TFA/TIS/H2O (95/ spectroscopy 2.5/2.5, (v/v/v)) mixture for 2 h at room temperature. The resin was filtered out and washed with TFA. Filtrate and TFA washes were EPR measurements were carried out on a BRUKER combined and TFA removed under a nitrogen stream. The final ELEXSYS 500 X-band CW-ESR spectrometer, equipped with a peptides were precipitated in cold Et2O, recovered by centrifugation, BVT 3000 digital temperature controller. The spectra were dissolved in water and lyophilized. recorded at 120 K in frozen DMSO solutions otherwise Frontiers in Pharmacology 04 frontiersin.org Peña et al. 10.3389/fphar.2022.1060827 noticed. Typical acquisition parameters were as follows: Cell-viability assays microwave power 10–20 mW, modulation frequency 100 kHz, modulation gain 3 G. Simulations were performed using the HeLa and MFC7 cells were seeded into a 96-well plate at a cell EasySpin toolbox developed for MATLAB (Stoll and density of 3·103 cells/well in 100 µL of culture medium and Schweiger, 2006). allowed to grow overnight. The next day, the C1-C5 complexes were added to cells at a range concentration of 0–200 μM. Working concentrations of complexes C1-C5 Ultraviolet-visible (UV-vis) spectroscopy (<0.1% DMSO in biological experiments, required for C1) were prepared in DMEM (for HeLa) and DMEM/F-12 (for All the spectra were recorded at room temperature either on MCF7) medium. The growth inhibitory effect of the an Agilent HP 8453, a Varian Cary 50 Bio, a Varian Cary 60 Bio complexes was measured at 24 h using the PrestoBlue Cell or a Perkin Elmer Lambda 650 spectrophotometer, using 1 cm Reagent (Life Technologies) assay. Briefly, PrestoBlue (10 μL; quart-cuvettes. Ascorbate consumption experiments were resazurin-based solution) was added to each well. After 2 h monitored by UV-vis at the maximum absorption band of the incubation (37°C, 5% CO2), the fluorescence of each well was ascorbic acid (100 µM) at 265 nm for about 45 min. CuCl2 and measured at 572 nm after excitation at 531 nm with a Microplate the assayed complexes C1-C3 were added at a final concentration Reader Victor3 (Perkin Elmer). Cytotoxicity was evaluated by of 2 µM in 50 mM NaCl/5 mM TRIS-HCl buffer (pH 7.2), with a relative cell viability (%) for each sample related to the control maximum of 5% of DMSO. well. The IC50 values were calculated from the obtained cell viability results. Each complex was tested per triplicate and averaged from three independent sets of experiments. Blank Inductively coupled plasma (ICP) optical and complex controls were also considered. For experiments emission spectrometry (OES) and mass of C1 and C5 at 30 min and 4 h of treatment, cells were plated and spectrometry (MS) treated following the same protocol. After each treatment time, the supernatant was removed, cells were washed, and fresh media ICP-MS was performed on an Agilent apparatus, model was added. Cells were allowed then to evolve until a total 7500ce. ICP-OES was carried out in a Perkin-Elmer, model incubation time of 72 h and the cell viability was measured Optima 4300DV. Both used standard acquisition parameters with PrestoBlue®, as beforehand detailed. for copper content. Cellular copper uptake studies Stock solutions of complexes C1-C5 for biological assays HeLa and MCF7 cells were plated, grown, and allowed to adhere overnight in a 6-well plate (2·105 cells/well). Cells were treated for 4 h Stock solutions of the assayed complexes C1-C5 were with the different copper complexes at the desired concentration. prepared by weighing the appropriate amount of complex Before analysis, medium was removed, and cells were washed with and diluting them in the corresponding solvent (DMSO for DPBS (Dulbecco’s Phosphate Buffered Saline, Invitrogen) and C1, and H2O for C2-C5). Quantification of the copper trypsinized for 10 min. The samples were harvested by concentration was carried out at the Servei d’Anàlisi centrifugation (1,400 rpm, 5 min) and the cellular pellets were Química (UAB) by ICP-OES. Measurements were collected and digested with concentrated HNO3. Quantification performed at least per duplicate. of the intracellular copper was performed by using inductively coupled plasma mass spectrometer (ICP-MS). Measurements were performed at least per duplicate. Cell cultures Human cancer cells (cervix: HeLa; and breast: MCF7) were Intracellular ROS production assays obtained from American Type Culture Collection (ATCC, Manassas, VA, United States). Cells were routinely cultured HeLa and MCF7 cells were plated and allowed to adhere with DMEM (Dulbecco’s modified Eagle’s medium, overnight in a 96-wells plate (2·104 cells/well). The 2′,7′- Invitrogen) for HeLa, and with DMEM/F-12 (Dulbecco’s dichlorofluorescin diacetate reagent (DCFDA, 25 μM in MEM/Nutrient Mixture F-12 Ham, Invitrogen) for MCF7, DMSO) was then added and the cells incubated at 37 °C in both supplemented with 10% heat-inactivated fetal bovine the dark for 30 min. The DCFDA solution was removed and cells serum (FBS, Invitrogen) at 37°C in a humidified 5% CO2 were treated with C1 and C5 at the corresponding IC50 values (at atmosphere. 24 h) and incubated for 4 and 24 h. The experiments were run in Frontiers in Pharmacology 05 frontiersin.org Peña et al. 10.3389/fphar.2022.1060827 SCHEME 1 Synthetic procedures to obtain H2L1 functionalized ligands (H2L2-H2L5). (A) Condensation reaction to obtain ligands H2L2-H2L5 from the corresponding precursors 3-6. Arginine in 4, peptide sequences in 5 and 6, and H2L3-H2L5 have an amide group instead of a carboxylic group at the C-terminal site. (B) Schematic synthetic procedure to obtain salicylaldehyde functionalized precursors 4-6. Arginine side-chain in peptides 11 and 12 (R9*) have a 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) protecting group (PG). Colored in blue, the amine terminal functional group employed to form the amide bond with the carboxylic acid (-COOH, in red) following standard HBTU coupling procedure. TFA refers to trifluoroacetic acid. triplicate. H2O2 was used as a positive control at 100 µM. The The plate was washed three times with PBS-BSA 0.5% between every fluorescence of each well was measured at 535 nm with a step. The remaining binding sites were blocked with PBS-BSA 0.5% Microplate Reader Victor3 (Perkin Elmer) after excitation at for 2 h. The antiserum samples were serial diluted at a range 485 nm. concentration of 1/10–1/10,000 (v/v). The samples were applied in triplicate and incubated for 1 h at room temperature. HRP (horseradish peroxidase)-conjugated secondary antibody anti- Antibody production and titer rabbit (Bio-Rad) diluted in blocking buffer was incubated with the samples for 1 h at room temperature. TMB (3,3′,5,5′- Rabbit Poly-Arginine (9R) antibody was produced by Davids tetramethylbenzidine, ThermoFisher) solution was added to each Biotechnologie GmbH (Germany) as follows. New Zealand white well and incubated for 30 min for signal detection. An equal volume rabbits were immunized with newly synthesized Acetyl- of stopping solution (2 M H2SO4) was added and the completed RRRRRRRRR-Amide peptide and the antiserum was further reaction was read at 450 nm in a microplate reader (Tecan). purified by affinity purification. The obtained 9R antibody (Anti- R9) was tittered by means of ELISA (enzyme linked immunosorbent assay) to determine the optimal dilution for immunocytochemical Immunofluorescence assays detection. Briefly, MaxiSorp (Sigma-Aldrich, MO, United States) microtiter plate was coated with the Acetyl-RRRRRRRRR-Amide HeLa cells were seeded in 24-well plates at a cell density of 5·104 peptide in carbonate/bicarbonate buffer (pH 9.6) overnight at 4°C. cells/well in 1 ml of culture medium and allowed to grow overnight on Frontiers in Pharmacology 06 frontiersin.org Peña et al. 10.3389/fphar.2022.1060827 fluorescence and confocal microscopy. Images were captured using a Leica TCS SP5 laser scanning confocal microscope with a PL APO 63×/1.4-0.6 Oil objective and processed using Imaris software. Statistical analysis Results are presented as means ± standard deviation in all figures and in Table 2. Each viability assay determination experiment was performed in three independent experiments, while each confocal microscopy and cellular uptake experiment was performed in at least two independent experiments. GraphPad Prism was used for graphs and associated calculations. Results FIGURE 2 Synthesis and characterization of the Cu(II) coordination environment of complexes C1-C5. X-EPR band spectra of complexes C1-C5 in frozen DMSO solution at ligands H2L2-H2L5 and the corresponding 120 K, confirming the same Cu(II) coordination environment in all copper(II) complexes the complexes. Ligand H2L1 and its corresponding complex C1 were synthesized as previously reported (Peña et al., 2021). The four newly synthesized ligands H2L2-H2L5 were essentially prepared by imine bond a sterilized coverslip. The next day, cells were incubated with 50 µM of formation between the monoprotected benzene-1,2-diamine (1) C5 or control peptide (R9) for 4 h. For cell fixation, culture medium and the corresponding salicylaldehyde precursor (3-6, Scheme 1A). was removed, cells were washed with DPBS and fixed with 4% The synthesis of the different ligands and their respective precursors, paraformaldehyde (PFA, Sigma-Aldrich) at room temperature for including the corresponding characterization data, can be found in the 20 min. PFA was then removed and cells were washed and blocked experimental section and as part of the Supplementary Material with PBS containing 3% BSA (bovine serum albumin, Sigma-Aldrich) (Supplementary Figures S1-9). and 0.1% Triton X-100 (Sigma-Aldrich) for 30 min. Cells were then For H2L2, aldehyde (3) was achieved through a para-sulfonation of incubated overnight at 4 °C with Anti-R9 antibody (Rabbit, 3.4 μg/ml) the starting material 2-hydroxybenzaldehyde (2), via an electrophilic and anti-LAMP-1 (Rat, 2 μg/ml, Santa Cruz Biotechnology, CA, aromatic substitution (SEAr) reaction (Clayden et al., 2012). Standard United States). Polyclonal IgG anti-rabbit Alexa 568, and anti-rat sulfonation reactions are normally performed at temperatures around Alexa 488 antibody (1:500, Thermo Scientific) were added after 100°C, however, this step was carried out at 40°C to prevent significant removal of the primary antibodies and washing cells three times oxidation of the aldehyde in such acidic conditions (Kirker, 1980). with PBS (1% BSA and 0.1% Triton X-100). Nuclei and cell Regarding the synthesis of the salicylaldehyde precursors of the ligands membrane were counterstained with 4’,6-diamidino-2-phenylindole H2L3-H2L5 (compounds 4–6), similar procedures were followed for all (DAPI) (1:500, 5 min) and Alexa-633 conjugate of wheat germ three, namely attaching the Arg residue (H2L3, precursor 4) or the agglutinin (WGA) (2 μg/ml, 10 min, Thermo Scientific) corresponding CPPs (H2L4-H2L5, precursors 5 and 6, respectively) to respectively. The slides were mounted and prepared for the common intermediate 9 through a stable amide linkage via TABLE 1 EPR parameters from X-EPR band spectra of complexes C1-C5. Spectra were recorded at 120 K in frozen DMSO solutions. gz/Az ratio is a parameter that predicts the distortion from planarity of the structure (gz/Az > 140 cm to consider a planar distortion in the structure). Complex gz Az (10−4 cm−1) gx/gy Ax Ay gz/Az (cm) (10−4 cm−1) C1 2.244 183 2.043/2.076 <30 123 C2 2.246 183 2.040/2.074 <30 123 C3 2.247 184 2.045/2.071 <30 122 C4 2.247 185 2.041/2.074 <30 121 C5 2.247 185 2.047/2.075 <30 121 Frontiers in Pharmacology 07 frontiersin.org Peña et al. 10.3389/fphar.2022.1060827 the same procedure as previously described for C1 (Peña et al., 2021). The complexes were isolated from the reaction media by precipitation. Experimental evidence of Cu(II) complexation was initially obtained for the complexes with the two simplest modifications (i.e., C2 and C3) by the appearance of metal-to- ligand or ligand-to-metal charge transfer bands (MLCT, λ~ 420 nm), and Cu(II) d-d bands (λ~ 650 nm) in the ultraviolet- visible (UV-vis) spectra (Supplementary Figure S10). Additionally, the electronic transitions observed in the functionalized complexes are analogous to those obtained for C1, outlining that the chemical derivatization did not affect the coordination capabilities of the ligands. The presence of the molecular peaks for C2-C5 in high- resolution mass spectrometry (HR-MS, Supplementary Figure S11) FIGURE 3 corroborated the successful metal coordination in all cases. Confirmation of reactive oxygen species (ROS)-generation capabilities after functionalization using the ascorbate Comparison of the EPR spectroscopic features and calculated consumption experiment. Ascorbic acid (100 µM) consumption of characteristic parameters (g and A tensors) of C2-C5 with those of CuCl2, C1, C2 and C3 (2 µM) monitored by UV-vis the parent complex C1 (Figure 2; Table 1) confirmed that the newly spectroscopy at 265 nm, in 50 mM NaCl/5 mM TRIS-HCl buffer at pH 7.2. synthesized complexes do maintain the same Cu(II) coordination environment as C1, with the metal center located in a N2O2 in-plane coordination environment (Peisach and Blumberg, 1974) and with the unpaired electron in a dx2 −y2 orbital (gz > gx,y > ge), thus adopting TABLE 2 IC50 (µM) values at 24 h of complexes C1-C5 in HeLa and a square-planar or square-pyramidal derived geometry. The MCF7 cell cultures, normalized for copper (Cu) concentration. The results shown are representative of at least three independent calculated gz/Az ratio is lower than 140 cm for all of them, which experiments (N ≥ 3). is indicative of a non- or marginally-distorted structure from planarity (Sakaguchi and Addison, 1979). Consequently, and Compound HeLa MCF7 taking all the above insights together, the different C1 67 ± 2 32 ± 16 functionalization strategies assayed have not altered the metallic C2 112 ± 4 14 ± 9 core of C1, present in all new complexes C2-C5. C3 107 ± 7 8±3 C4 51 ± 6 1.1 ± 0.6 C5 50 ± 5 0.6 ± 0.4 Effect of functionalization on the copper- CuCl2 ≥ 200 ≥ 200 mediated ROS production capabilities Prior to biological evaluation, and after proving that the new standard solid-phase Fmoc-methodologies (Scheme 1B) (Chan and C2-C5 Cu(II) complexes hold the same metallic core as C1, the White, 2000). impact of the ligand substituents on their catalytic properties, A Rink amide resin was chosen in all the cases to obtain an and therefore, ROS-generating capabilities (Peña et al., 2021), amide functional group in the C-terminal position of the Arg or were confirmed with the two simplest functionalized Cu(II) CPP scaffold, in order to avoid competition in the Cu(II) compounds (C2 and C3), as model compounds bearing complexation step (vide infra). Deprotection and cleavage oppositely-charged functional moieties. The consumption of steps from the resin were performed using standard methods, ascorbate was measured by UV-vis spectroscopy, monitoring and preparative reversed-phase HPLC purification rendered the its characteristic absorbance at 265 nm. The correlation of pure salicylaldehyde precursors 4-6. To note, the optimized ascorbate consumption with the generation of ROS has strategy to synthesize the precursors 4-6 required the use of proven to be a reliable method to effectively monitor ROS the imine intermediate benzoic acid 9, which was obtained by production (Alies et al., 2013; Chassaing et al., 2013). condensation reaction between the MOM-hydroxyl protected In the presence of ascorbate and in an aerobic environment, compound 8 and the monoprotected diamine 1. The phenol Cu(II) catalyzes the generation of ROS upon ascorbate consumption protection with the MOM-group and the imine-bond formation (Halliwell and Gutteridge, 1990). Without copper (DMSO control), (the latter as a strategy to mask the aldehyde group) were crucial no decrease in the absorbance at 265 nm was observed (Figure 3). In to increase the yield of the coupling reactions between 9 and 10- contrast, free Cu(II) ions (CuCl2) produced a rapid decrease in the 12, and to avoid the formation of by-products. absorbance and, after 20 min, the ascorbate was almost totally The Cu(II) complexes C2-C5 were obtained after metalation of consumed. For the sake of comparison, the ascorbate consumption the respective H2L2-H2L5 ligands with copper(II) acetate following capabilities of C1, C2 and C3 were examined at the same time and Frontiers in Pharmacology 08 frontiersin.org Peña et al. 10.3389/fphar.2022.1060827 FIGURE 4 In vitro cytotoxicity and cellular internalization of C1-C5 in HeLa and MCF7 cancer cells. (A) Cell-viability assays for complexes C1-C5 in HeLa and MCF7 cancer cell lines after 24 h treatment. (B) IC50 values for complexes C1-C5 after 24 h treatment, calculated from the corresponding cell- viability curves fitting (Figure 4A). (C) Quantification of the copper (Cu) uptake in HeLa and MCF7 cell lines after 4 h treatment with complexes C1-C5 (50 µM). (D) Correlation between the cytotoxicity (log (IC50)) and the Cu uptake for HeLa and MCF7, with the cell-penetrating peptide-bearing Cu(II) complexes circled. Concentrations of the complexes are normalized in all cases based on Cu concentration for comparison purposes. Statistical differences were determined using a one-way ANOVA test, with p values as: without symbol (p > 0.05, non-significant), *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Results average at least three independent experiments (N ≥ 3) for IC50 values, and two (N ≥ 2) for the Cu uptake. Statistical analysis and goodness-of-fit for linear regression (r2) using alpha threshold of 0.05 were calculated using GraphPadPrism. conditions. C3 consumed ascorbate at similar rates to C1, and just elucidate the effect of the different derivatizations. All the C2-C5 slightly slower rates were observed for C2 (Figure 3), with total synthesized complexes are soluble in water (at least 5 mg/ml for C2- consumption after 25 min in all cases. C3, and 20 mg/ml for C4-C5) and in biological medium, without the need of any percentage of DMSO (as it was required for the biological evaluation of C1 because of solubility issues). The Cytotoxicity assays and intracellular dose-response curves are shown in Figure 4A, and the obtained copper(II) accumulation IC50 values are plotted in Figure 4B and summarized in Table 2. To ensure comparability, all the stock solutions of C1-C5 were In vitro assays with the complexes C2-C5 were carried out in standardized by ICP-MS prior to biological evaluation and results HeLa and MCF7 cancer cells in order to (1) compare their activity normalized based on the copper content. with that of C1 (Figure 4), which we previously reported to be As beforehand mentioned, the first derivatization strategy cytotoxic against several cell lines (Peña et al., 2021), and to (2) (sulfonate and Arg) mainly aimed at increasing the solubility of Frontiers in Pharmacology 09 frontiersin.org Peña et al. 10.3389/fphar.2022.1060827 Table 2), thereby suggesting that the intracellular delivery plays a key role in the final activity. Thus, cellular uptake studies were carried out to deepen into the impact of the tailored modifications on the internalization of the five complexes C1-C5. ICP-MS was used to quantify the amount of Cu(II) inside cells (Figure 4C). Intracellular copper content after treatment with complexes C1-C3 was below 150 ng/106 cells, with similar uptake values among the three. Importantly, while sulfonate and Arg derivatizations, with the concomitant increase in solubility of the corresponding C2 and C3 complexes, did not mediate copper uptake, functionalization with CPPs (C4 and C5) significantly promoted intracellular copper delivery (Figure 4C). The amount of metal able to accumulate inside cells for the CPP-conjugated complexes is at least about twofold the amount observed for C1 (272 ± 15 and 315 ± 6 versus 131 ± 21 ng/106 cells in HeLa, and 178 ± 2 and 156 ± 22 versus 88 ± 2 ng/106 cells in MCF7, for C4 and C5 versus C1, respectively). Based on the data, the presence of the G4 linker in C5, FIGURE 5 which was designed to provide higher conformational flexibility (and In vitro reactive oxygen species (ROS) production. ideally better interaction of the peptide with the cell membrane), did Intracellular levels of ROS, measured with the DCFDA assay in not result in clear differences in uptake or cytotoxicity as compared to HeLa and MCF7 cancer cells for complex C1 and C5 at their IC50, 24h values and two different times (4 and 24 h). H2O2 (100 µM) C4 (at least in the assayed cancer cell lines thus far). was used as positive control. The values are plotted in percentage Although there are multiple factors influencing the (re)activity respect to cells control, which is 100%. of metal complexes (both from chemical and biological features), we attempted to evaluate how strongly the intracellular delivery is related to the anticancer activity of these compounds. Thus, we correlated the obtained IC50 values (in log) with the copper uptake the C2 and C3 complexes respect to C1. Results underline that, (intracellular copper amount). As exemplified in Figure 4D, this although improved aqueous solubility was attained, none of the resulted in a very good correlation for HeLa cancer cells (with r2 = two modifications contribute to enhance the cytotoxicity of the 0.9 and p value of 0.02). For MCF7, the correlation was still fairly parent complex in cancer cells, with IC50 values higher than (in good (r2 = 0.7) and, despite not being strictly significant by definition HeLa), and in the range of (in MCF7) complex C1 (Figure 4B). In (p value of 0.08), there is a clear tendency outlining intracellular contrast, functionalization with the CPPs (complexes C4 and C5) copper delivery as an important design parameter to modulate resulted in improved cytotoxicity with significantly lower IC50 cytotoxicity in these (and, by extension, likely in similar) Cu(II) values, particularly in MCF7 (IC50,C1-to-IC50,C4-C5 ratio ≥30, complexes. FIGURE 6 Cellular localization and cytotoxicity efficacy of cell-penetrating peptide-conjugated Cu(II) complex C5. (A) Cell-viability assays for complexes C1 and C5 (100 µM) in HeLa and MCF7 cell lines at 30 min or 4 h treatment times, after which the cell media was replaced by fresh medium and cells incubated for extra time until reaching a total of 72 h. Statistical differences were determined using a one-way ANOVA, with p values as: ****p ≤ 0.0001. (B) Cellular distribution of R9 peptide (control experiment) and complex C5 after 4 h incubation in HeLa cancer cells. HeLa cells were fixed and stained with Anti-R9 (red) and anti-LAMP-1 (green, for lysosomes) antibodies, and DAPI (blue, nuclei). Last column shows the merged images. Frontiers in Pharmacology 10 frontiersin.org Peña et al. 10.3389/fphar.2022.1060827 Effect of cell-penetrating peptide- their inherent cellular distribution. This is basically because conjugation: Reactive oxygen species, fluorophores are generally hydrophobic and they can change efficiency and cellular distribution the solubility, flexibility and conformation of the final CPP functionalized complexes (Szeto et al., 2005; Puckett and To gain more insights into the behavior of the CPP- Barton, 2009; Mishra et al., 2011; Hirose et al., 2012). Thus, in bearing Cu(II) complexes, and taking into account the order to avoid interference or misleading conclusions, we used similar profile exhibited by C4 and C5 (with slightly higher immunofluorescence with an Anti-R9 antibody (specifically toxicity for the latter), C5 was selected for further comparative produced with a R9 peptide as antigen; Acetyl-R9) to provide evaluation with C1. We initially corroborated the in vitro ROS an initial proof-of-concept of the intracellular distribution of production capabilities of C5 using the DCFDA assay complex C5 in HeLa cells (Figure 6B). (Figure 5). Data confirm that the CPP-conjugated Cu(II) Analysis with confocal microscopy after 4 h incubation time complex at their IC50 value was successfully able to revealed a diffused distribution throughout the cytosol produce significant levels of ROS in both tested cancer cell compartment, with few punctual accumulations in the nuclei. lines, reaching similar values to the positive control H2O2 These results demonstrate that C5 was able to accumulate inside (especially after 24 h incubation) and, thus, reinforcing the cells after 4 h, which is in concordance with what was observed in ROS-mediated pathway as an important mechanism of action the copper uptake experiments (Figure 4C). In contrast, for this set of complexes. It is also worth mentioning that, incubation with the Arg-rich CPP (R9 peptide, Figure 6B), as especially in MCF7 cancer cells, complex C5 was able to a control, showed localization in both the cytoplasm and the trigger the generation of a similar level of ROS to C1 yet nuclei, with preferential accumulation in the nuclei, where it using 50-fold less amount of complex, in line with the remains most-likely retained partly due to the interaction with observed enhanced uptake and higher cytotoxicity of C5. the negatively-charged phosphate backbone of the DNA. In order We then set out to evaluate the impact of the CPP-mediated to elucidate whether the linker modulates subcellular internalization in the cytotoxic efficacy of the final complex. To this localization, we then also carried out immunofluorescence end, cytotoxicity assays were performed at shorter treatment time assays with C4. As observed from the confocal microscopy for C1 and C5 (4 h, Figure 6A). After that specific incubation time, images (Supplementary Figure S12), the intracellular the supernatant of the cells was removed (containing the non- distribution of C4 was analog to that of C5 (i.e., mostly internalized complex), cells were washed, new cell culture medium diffusively localized in the cytosol), thus overall suggesting added, and cells were incubated to complete 72 h of total incubation that the linker does not have a clear impact on intracellular time. Data show that whilst there is almost no cell-death for C1, distribution either. Promisingly, both C4 and C5 results show no C5 drastically reduced the cell viability to more than 60% in HeLa colocalization of the CPP-conjugates in the lysosomes after 4 h and almost 100% in MCF7 after 4 h treatment (Figure 6A). These incubation which, together with the diffused distribution pattern results reveal that the presence of the CPP not only promoted in the cytosol, could indicate potential endo-/lysosomal bypass intracellular delivery, but, consequently, it also mediated a faster and/or escape. anticancer response, understood as a significantly higher activity than the parent complex C1 at short incubation times. The increased cytotoxicity-to-time ratio observed for C5 would contribute to Discussion enhance therapeutic efficiency, also potentially minimizing the extent and impact of clearance and drug resistance pathways of Metals and their corresponding complexes have already tumoral cells due to the faster action. shown remarkable contributions in oncology, both in diagnosis Finally, we studied the subcellular localization of C5 in (e.g., gadolinium or technetium) and therapy (e.g., Pt) (Mjos comparison to the CPP moiety alone in order to elucidate the and Orvig, 2014; Anthony et al., 2020). Despite the significant fate of the conjugated cargo inside the cells. Several biophysical side-effects encountered, the three worldwide approved Pt- methods have been traditionally used to assess the cellular drugs (cisplatin, carboplatin and oxaliplatin) are still distribution of CPPs and their cargoes (e.g., fluorescence, administered to at least 20% of cancer patients nowadays, radiolabeling, Raman spectroscopy and X-Ray scattering), and they represent first-line treatments for testicular, being those based on fluorescence the most extensively used ovarian, bladder and colorectal cancer, among others, thus (Bechara and Sagan, 2013). Usually, peptides are covalently underlining the (still fully unlocked) potential of metal-based coupled to a fluorophore, and monitoring of the fluorescence anticancer therapy (Casini et al., 2019; Rottenberg et al., 2021; allows for cellular localization with confocal microscopy. Peña et al., 2022). The recent years have evidenced that However, besides all the experimental factors that may complexes based on physiological (catalytic) metals that act influence the uptake mechanism and subsequent localization via different mechanisms of action, like ROS generation, can (e.g., cell type, incubation time and temperature), the offer promising therapeutic responses (Li et al., 2020; Liu et al., functionalization of CPPs with dyes has been reported to alter 2019; Lovejoy et al., 2011; Luo et al., 2014; Shen et al., 2020; Frontiers in Pharmacology 11 frontiersin.org Peña et al. 10.3389/fphar.2022.1060827 Trachootham et al., 2009; Zhang and Sadler, 2017). properties like solubility, (in vitro) ROS-generation capabilities, Additionally, several studies emphasize the relevance of cytotoxicity, intracellular metal delivery and subcellular distribution. ROS-based cancer therapy as one of the strategies to avoid For that purpose, ligand H2L1 was functionalized with a and/or bypass Pt-induced resistance while increasing sulfonate group (H2L2) and an Arg residue (H2L3), with the therapeutic selectivity towards cancer cells (Cui et al., 2018; main goal of increasing solubility, and with two variants of a Wang et al., 2021). nona-Arg CPP (Guidotti et al., 2017) (without linker, H2L4, and In this context, Cu(II) complexes have exhibited potential as with, H2L5), for both solubility and intracellular delivery. Our data anticancer agents (Santini et al., 2014). They can combine both confirmed that the ligand scaffold was successfully tailored with the features: they encompass a (more) biocompatible metal center different functionalities, being the two variants of CPPs (for and the possibility of triggering ROS production, as we and H2L4 and H2L5) linked through an analog chemical procedure others have recently reported (Zubair et al., 2013; Ng et al., 2014; as that carried out for the single Arg residue (H2L3). Noteworthy, Sîrbu et al., 2017; Peña et al., 2019, 2021). However, certain conjugating Arg and Arg-rich CPPs to the ligand scaffold via the physicochemical properties can limit the anticancer activity and same chemical protocols outlines the robustness and versatility of hamper further (pre)clinical evaluation of newly developed such derivatization approach, which can be then analogously (metal) anticancer agents. One crucial aspect relies on the lack extended to future tailoring of this and similar (salphen-based) of adequate solubility in biological fluids (Savjani et al., 2012) ligands and metal complexes with other peptides or cancer-targeting (about 90% of preclinical drug candidates present low-water moieties containing amine functional groups. All the resulting solubility (Kalepu and Nekkanti, 2015)), as was for instance Cu(II) complexes (C2-C5) exhibited improved water solubility. observed for our recently reported ROS-producing cytotoxic Given that the cytotoxic activity of the Cu(II) complex C1 is Cu(II) complex C1 (Peña et al., 2021). A second key strongly linked to ROS production (Peña et al., 2021), we specifically parameter in (metallo)drug discovery is based on the capacity designed the functionalization strategies to be located on the to cross (cellular) membranes (which is related to properties such periphery positions of the ligand H2L1, distant to the Cu(II) as lipophilicity) (Vargason et al., 2021). coordinating atoms to minimize alterations of the metallic core Over the years medicinal chemistry has explored and and preserve its ROS generation capabilities. Cytotoxicity assays in established multiple strategies to overcome such limitations HeLa and MCF7 cancer cells highlighted that while the and efficiently deliver chemotherapeutics; many of them functionalization with sulfonate (C2) or Arg (C3) groups (i.e., involving structural modifications like adding pH-sensitive modifications only targeting solubility issues) did not represent groups that enhance aqueous solubility and conjugation to any improvement in the final in vitro anticancer activity, the CPPs, among others. CPPs are typically made up of conjugation to CPPs (i.e., complexes C4 and C5) promoted 5–30 amino acids, and they can be utilized as molecular higher, faster and, therefore, enhanced ROS-mediated anticancer transporters to facilitate the passage of therapeutic drugs activity respect to C1; overall in line with the increase in intracellular across physiological barriers (Copolovici et al., 2014; Guidotti copper levels. The differences observed in copper uptake can be thus et al., 2017). Up to now, (cationic Arg-rich) CPPs have been directly attributed to the presence of the CPPs, which actively widely used in many anticancer treatment strategies, successfully mediate the cellular internalization of C4 and C5 and, addressing both solubility and intracellular delivery issues of a correspondingly, impact on their cytotoxicity. variety of anticancer therapeutic molecules (Zhou et al., 2022). Importantly, we confirmed that the presence of only one Arg Our goal in this work was to improve the anticancer activity of residue (C3) is not sufficient to enhance cellular uptake despite the promising ROS-producing Cu(II) complex C1 by employing improving aqueous solubility and, by extension, bioavailability. In solubility- and/or uptake-targeting ligand modifications like CPP- some cases, it even represented a drawback regarding intracellular conjugation. However, there is still a lack of rationalization and copper delivery and cytotoxicity (e.g., in HeLa cancer cells). These understanding of how structural features and chemical results highlight that specific solubility-targeting derivatizations do modifications can influence the activity of the final compound, not correlate with an enhanced anticancer activity, and that they especially in metal-containing systems. Different from purely might indeed negatively impact other key drug properties such as organic-based compounds, the (re)activity of metal-based cellular membrane penetrating capacity. All in all, the conjugation structures is not only dependent on the metal center (and its with (Arg-rich) CPPs can target and maximize both properties at oxidation state) or on the ligand scaffold, but also (and arguably once, thus underlining the importance of the adequate choice of even more) on the metal-ligand interaction (Anthony et al., 2020; chemical modifications in a more rational and holistic manner. Peña et al., 2022). Consequently, systematically and comparatively Further systematic evaluation of these (and similar CPP-conjugated) exploring several of such relevant and widely used derivatizations, metal complexes regarding biocompatibility and therapeutic especially CPP-conjugation, enabled us to deepen, at the same time, performance in healthy primary cells and in vivo models would into structure-behavior relationships of Cu-based anticancer agents provide further insights into the added value of CPP-conjugation from different aspects, mostly encompassing physicochemical strategies in the development of metal-based anticancer agents. Frontiers in Pharmacology 12 frontiersin.org Peña et al. 10.3389/fphar.2022.1060827 Finally, we set out to explore the subcellular distribution of the Author contributions CPP-conjugates as compared to the free peptide. For both C4 (without linker) and C5 (with linker), the ubiquitous intracellular QP, PB, MC, OP, JL, and OI conceived the work. QP, SR-C, distribution in the cytosol, together with the absence of specific and AS performed the experiments. QP, SR-C, JL, and OI wrote lysosomal co-localization, might suggest non-endosomal uptake or the first draft of the manuscript. AS, PB, MC, and OP provided rapid endosomal entrapment followed by endosomal release comments and corrections to the manuscript draft. All the (Guterstam et al., 2009; Liu et al., 2018). One of the commonly authors have read and approved the final version. reported intracellular delivery pathways of cell-penetrating peptides relies on endocytosis-like mechanisms. This generally results in the entrapment of the peptides and their cargo, (i.e., the Funding Cu(II) complexes), inside the endosome and, subsequently, lysosomal compartments, whose low pH (4–5) and high This work was supported by the Spanish Ministerio de enzymatic activity can degrade the compound and, hence, Ciencia e Innovación and FEDER to the projects BIO2015- impair its activity (Lecher et al., 2017; Pei and Buyanova, 2019; 67358-C2-2-P, CTQ2015-70371-REDT, RTI2018-098027-B- Kondow-Mcconaghy et al., 2020). Nonetheless, in other cases (e.g., C22 and PID2021-127983OB-C22. for specific sequences of Arg-rich CPPs, certain cargoes, type of cell and at determined concentrations), other mechanisms involving (passive) direct translocation or fusion pore formation have also Acknowledgments been relevantly proposed (Brock, 2014; Allolio et al., 2018). Although it is still complex to univocally elucidate the The Servei d’Anàlisi Química (UAB) and Spectropole facilities underlying entry pathways for CPP-conjugates, the comparison (AMU) are kindly acknowledged for allocating instrument time. of the different distribution profiles between the R9 peptide alone Alejandro Garcia Torrado is acknowledged for his contributions and both C4 and C5 corroborates that the attachment of the CPP to the synthesis and characterization of the H2L2 ligand and the to the Cu(II) complex has altered the intracellular biodistribution C2 complex. Some of the figures were partly designed using pattern. Additionally, the overall data suggest that the presence of BioRender® software. QP and SR-C acknowledge the Ministerio the linker has no apparent effect in the intracellular distribution de Educación, Cultura y Deporte the financial support of an FPU either, in line with the similar cytotoxicity and intracellular copper grant. The authors from the UAB are members of the “Grup de levels observed for both C4 and C5. It is undeniable that further Recerca de la Generalitat de Catalunya” ref. 2017SGR-864 and elucidation of the molecular details of the entry and endosomal 2017SGR-1584. escape pathways, especially when involving metal-based compounds, are required to strengthen (metallodrug) structure- function relationships (Brock, 2014). Conflict of interest In conclusion, our data suggest that intracellular copper delivery plays an important role in governing the final The authors declare that the research was conducted in the cytotoxicity of redox-active Cu(II) complexes, while only absence of any commercial or financial relationships that could tuning solubility by adding (positively/negatively) charged be construed as a potential conflict of interest. groups does not translate into an increased cytotoxic activity, and may also hinder intracellular delivery and hamper crossing (cellular) membranes. Beyond the therapeutic potential shown Publisher’s note by the CPPs-conjugated complexes (C4 and C5), we expect that the systematic evaluation and understanding about the role of the All claims expressed in this article are solely those of the different tailored modifications contributes to (1) enhance the authors and do not necessarily represent those of their affiliated value of (Arg-rich) CPP-conjugation in (metallo)drug discovery, organizations, or those of the publisher, the editors and the and to (2) optimize the design of these and similar (redox-active) reviewers. Any product that may be evaluated in this article, or metal complexes for enhanced and faster intracellular delivery claim that may be made by its manufacturer, is not guaranteed or and, consequently, improved anticancer activity. endorsed by the publisher. Data availability statement Supplementary material The original contributions presented in the study are The Supplementary Material for this article can be found included in the article/Supplementary Material, further online at: https://www.frontiersin.org/articles/10.3389/fphar. inquiries can be directed to the corresponding authors. 2022.1060827/full#supplementary-material Frontiers in Pharmacology 13 frontiersin.org Peña et al. 10.3389/fphar.2022.1060827 References Alies, B., Sasaki, I., Proux, O., Sayen, S., Guillon, E., Faller, P., et al. (2013). Zn leads to their direct penetration into cells. Mol. Ther. 20 (5), 984–993. doi:10.1038/mt. impacts Cu coordination to amyloid-β, the alzheimer’s peptide, but not the ROS 2011.313 production and the associated cell toxicity. Chem. Commun. 49 (12), 1214–1216. Kalepu, S., and Nekkanti, V. (2015). Insoluble drug delivery strategies: review of doi:10.1039/c2cc38236a recent advances and business prospects. Acta Pharm. Sin. 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