molecules Article Assessing Amphiphilic ABAB Zn(II) Phthalocyanines with Enhanced Photosensitization Abilities in In Vitro Photodynamic Therapy Studies Against Cancer Miguel Á. Revuelta-Maza 1 , Marta Mascaraque 1,2 , Patricia González-Jiménez 1 , Arturo González-Camuñas 1 , Santi Nonell 3 , Ángeles Juarranz 1,2, * , Gema de la Torre 1, * and Tomás Torres 1,4, * 1 Departments of Organic Chemistry and Biology, Universidad Autónoma de Madrid. C/Francisco Tomás y Valiente 7, 28049 Madrid, Spain; miguel.revuelta@uam.es (M.Á.R.-M.); marta.mascaraque@uam.es (M.M.); patri.gj.95@gmail.com (P.G.-J.); arturo.gonzalezc01@estudiante.uam.es (A.G.-C.) 2 Instituto Ramón y Cajal de Investigación Sanitaria, 28034 Madrid, Spain 3 Institut Químic de Sarrià. Universitat Ramon Llull, 08017 Barcelona, Spain; santi.nonell@iqs.url.edu 4 Instituto Madrileño de Estudios Avanzados (IMDEA)-Nanociencia. C/Faraday 9, Cantoblanco, 28049 Madrid, Spain * Correspondence: angeles.juarranz@uam.es (Á.J.); gema.delatorre@uam.es (G.d.l.T.); tomas.torres@uam.es (T.T.); Tel.: +34-91-4974151 (T.T.) Academic Editor: José A. S. Cavaleiro   Received: 1 December 2019; Accepted: 28 December 2019; Published: 4 January 2020 Abstract: We have previously demonstrated that singlet oxygen photosensitization abilities of Zn(II) phthalocyanines (Zn(II)Pcs) are enhanced through α-functionalization with bulky fluorinated substituents (i.e., bis(trifluoromethyl)phenyl units) at facing positions of ABAB Zn(II)Pcs, where A and B refer to differently functionalized isoindoles. In this work, we have prepared the Zn(II)Pc ABAB 1 endowed with hydrophilic triethylene glycol monomethyl ether (i.e., at the A isoindoles) to provide solubility in aqueous media, together with its A3 B and A4 counterparts, and compared their ability to behave as photosensitizers for photodynamic therapy. All photophysical data, aggregation studies and preliminary in vitro biological assays in cell cultures of SCC-13 (squamous cell carcinoma) and HeLa (cervical cancer cells), have proved ABAB 1 as the best photosensitizer of the series. Keywords: Zn(II) phthalocyanines; ABAB; amphiphilic; photodynamic therapy; HeLa; SCC-13 1. Introduction Phthalocyanines (Pcs) stand out for their chemical stability, versatility, and propensity to generate reactive oxygen species (ROS) when photoexcited with light of the far red region of the UV–Vis spectrum [1]. These properties make Pcs interesting for their application in medicine as photosensitizers (PS) for photodynamic therapy (PDT) against cancer [2]. Owing to their extended π-conjugation, Pcs usually exhibit high aggregation tendency and solvophobic effects, forming oligomers in solution [3,4]. This high level of aggregation affects their photochemical and photophysical properties, which results in their photodynamic inactivation, avoiding ROS and singlet oxygen (1 O2 ) generation and diffusion. Yet, aggregation tendency is not only their unique weakness, but also their inherent insolubility in water (which is crucial in order to reach the therapeutic target). These two facts together restrict drastically Pcs for in vivo application as PS. In this context, the choice of adequate substituents is decisive to achieve solubility in aqueous media and hinder their aggregation. Hydrophilic groups can be introduced to reach water solubility [5], whereas aggregation can be reduced or avoided by the introduction of bulky substituents in the Pc [6]. Among the different metallic Pcs used for PDT, Zn(II)Pcs stand out as one of the most promising, as they present high efficiencies in the generation of Molecules 2020, 25, 213; doi:10.3390/molecules25010213 www.mdpi.com/journal/molecules Molecules 2019, 24, x FOR PEER REVIEW 2 of 17 and hinder their aggregation. Hydrophilic groups can be introduced to reach water solubility [5], whereas aggregation can be reduced or avoided by the introduction of bulky substituents in the Pc [6]. Among Molecules 2020, the different metallic Pcs used for PDT, Zn(II)Pcs stand out as one of the most promising, 25, 213 2 of 18 as they present high efficiencies in the generation of 1O2 [7–10]. Recently, our research group has described new paradigmatic ABAB Zn(II)Pcs (with A and B coding for differently functionalized 1 O [7–10]. Recently, our research group has described new paradigmatic ABAB Zn(II)Pcs (with Ayieldand isoindole 2 constituents) that show both hindered aggregation and notably high 1O2 quantum (ΦΔ) values, featuring also chemical flexibility to incorporate additional functionalization that affords, B coding for differently functionalized isoindole constituents) that show both hindered aggregation and notably high 1 for instance, waterOsolubility 2 quantum [6].yield (Φ∆ )purpose, For that values, featuring also chemical precursor a bulky phthalonitrile flexibility endowed to incorporatewith additional functionalization that affords, for instance, water solubility bis(trifluoromethyl)phenyl units is used to avoid aggregation in the target ABAB Zn(II)Pc, but also [6]. For that purpose, a bulky phthalonitrile to hamper its precursor endowedand self-condensation, withthus bis(trifluoromethyl)phenyl force its cross-condensation units is used with to avoid other aggregation phthalonitrile in [11], the which target canABAB Zn(II)Pc, but with be functionalized also to hamper its groups. hydrophilic self-condensation, and thus force its cross-condensation with Theother use phthalonitrile of polyethylene glycol (PEG) chains iswith [11], which can be functionalized hydrophilic a popular groups. to enhance the approach The use of polyethylene biocompatibility of hydrophobic glycol (PEG) drugs inchains general is [12], a popular and ofapproach PDT agents to enhance the biocompatibility in particular. Among other of hydrophobic drugs in general [12], and of PDT agents in particular. advantages, PEG chains lead to a prolonged blood-circulating lifetime, allows for the minimization Among other advantages, PEG chains lead to a prolonged blood-circulating lifetime, of nonspecific uptake and favors the enhanced permeability and retention (EPR) effect. allows for the minimization of nonspecific All that uptake featuresand favors the combined canenhanced result in an permeability and retentionof(EPR) elevated concentration effect. the drug at All the that tumor features combined site [13]. From a can result in an elevated concentration of the drug at the tumor chemical point of view, PEG chains have proved to enhance the solubility in a variety of solvents,site [13]. From a chemical point of view, PEG chains have proved to enhance the solubility in a variety including nonpolar ones, aprotic polar solvents like DMSO, and protic polar solvents such as water of solvents, including nonpolar ones, [14,15]. aprotic Several polarPcssolvents like DMSO, functionalized with andPEGprotic chains polar solvents length of different such asaswater [14,15].[13,15–17] peripheral Several Pcs or functionalized with PEG chains of different length as peripheral [13,15–17] axial groups [18,19] have shown solubility in biological media, and some of them have been studied or axial groups [18,19] have shown as PS for solubility PDT [20,21]. in biological media, and some of them have been studied as PS for PDT [20,21]. Herein, we Herein, we report reportononthethe synthesis synthesis andand characterization characterization of a series of a series of triethylene of triethylene glycol glycol (TEG)- (TEG)-containing Zn(II)Pcs, namely ABAB-1, A B-1 and A -1, the study of their 1 O generation containing Zn(II)Pcs, namely ABAB-1, A3B-1 and3A4-1, the study 4 of their O2 generation2 capabilities, 1 capabilities, and preliminary and biological preliminary biological assays assays to to determine determine their potentialtheir as PSpotential for PDT.as ThePStarget for PDT.Pcs The havetarget been Pcs have been synthesized following two different synthetic pathways: synthesized following two different synthetic pathways: (i) a divergent route that allows to obtain (i) a divergent route that allows generictoABAB obtainand generic ABAB and A3B Zn(II)Pcs A3 B Zn(II)Pcswith functionalized functionalized phenolic groups with phenolic (ABAB-2groups and A3(ABAB-2 B-2 Zn(II)Pcsand A in3 B-2 SchemeZn(II)Pcs 1), whichin Scheme represent1), which synthons,represent synthons, over which it is over which possible it is possible to add hydrophilic to add hydrophilic groups such as groups PEG chains; such andas PEG chains; (ii) the and (ii) the preparation straightforward straightforward preparation of ABAB-1 and Aof 3B-1ABAB-1 (and also andAA 3 B-1 4-1) (and starting also 4 -1) starting fromAadequately from adequately functionalized functionalized phthalonitriles. Wephthalonitriles. have performedWe have performed aggregation aggregation and photophysical and photophysical studies in solution over this family of compounds studies in solution over this family of compounds in order to ratify our previous results regarding in order to ratify our previous results regarding the enhanced 1 O generation abilities of the nonaggregated ABAB-type Zn(II)Pc the enhanced O2 generation abilities 1 2 of the nonaggregated ABAB-type Zn(II)Pc compounds with compounds regard to their with A3regard B and A to4 counterparts their A3 B and[22]. A4 Going counterparts one step [22]. Goingweone further, have stepperformed further, we in have vitro performed in vitro assays to examine the toxicity assays to examine the toxicity of ABAB-1, A3B-1 and A4-1 in different of ABAB-1, A 3 B-1 and A tumor 4 -1 incells, i.e., SCC-13cells, different tumor and i.e., HeLa, SCC-13 as well andasHeLa, theirascell well as their cellThe localization. localization. final goalThe herefinal goal is to here is to demonstrate demonstrate the benefitsthe of benefits of bis(trifluoromethyl)phenyl-containing bis(trifluoromethyl)phenyl-containing Zn(II)Pcs versusZn(II)Pcsotherversus other substitution substitution patterns. patterns. Scheme 1. 1. Synthesis of triethylene glycol monomethyl monomethyl ether substituted substituted ABAB-1, ABAB-1, A A33B-1 and and A A44-1 ZnPcs. (i) (i)Zn(OAc) , o-DCB/DMF(2:1), Zn(OAc)2,2 o-DCB/DMF (2:1),150 150 ◦ ◦ °C,C, 1515 h; h; (ii)(ii) TEG-Ts, TEG-Ts, K2COK2 CO 3 , DMF, 3, DMF, 50 overnight; 50 °C, C, overnight; (iii) (iii) Zn(OAc) ◦ C, 1h. Zn(OAc) 2 , pentanol, 2, pentanol, 1,8-diazabicyclo 1,8-diazabicyclo [5.4.0]undec-7-ene [5.4.0]undec-7-ene (DBU), (DBU), 150 150 °C, 1h. Molecules 2020, 25, 213 3 of 18 2. Results and Discussion 2.1. Synthesis The synthesis of Pcs containing different substituents is usually carried out by cyclotetramerization of appropriately substituted precursors through the condensation of two types of phthalonitriles in a statistical process. Starting from the same proportion of both precursors, a mixture of six products is expected, but by using bulky phthalonitrile B [11] with two bis(trifluoromethyl)phenyl units, only three Zn(II)Pcs (i.e., ABAB, A3 B and A4 ) are usually obtained. For the preparation of our targeted Zn(II)Pcs, in particular ABAB-1 and A3 B-1, endowed with bulky groups over B isoindole rings and hydrophilic TEG substituents over the A positions, we have performed cross-condensation reactions with the bulky phthalonitrile B, and either A-1—already containing the TEG moieties—or 4,5-bis(4-hydroxyphenoxy)phthalonitrile A-2 [23] (see Scheme 1). The latter approach, yielding the intermediate Zn(II)Pcs ABAB-2 and A3 B-2 [24] that should be further O-alkylated, was tackled in the basis of previous studies, which had shown that the crossed condensation between B and A-2 is a highly efficient process (see below), and the Zn(II)Pcs formed are easily isolated from the reaction mixture [24]. Phthalonitrile A-1 was thus prepared by O-alkylation of A-2 [23] with the tosyl derivative of triethylene glycol monomethyl ether (TEG-Ts) [25] in 53% yield (see Scheme S1). Then, the reaction between phthalonitriles A and B was carried out by heating an equimolar mixture of them at 150 ◦ C in o-dichlorobenzene (o-DCB)/DMF and in the presence of Zn(OAc)2 . For the selected A:B ratio, the yield in A3 B Zn(II)Pcs is lower (8% for A3 B-1 and 7% for A3 B-2) than those obtained for their corresponding ABAB Zn(II)Pcs (10% for ABAB-1 and 17%, for ABAB-2). The O-alkylation reaction of ABAB-2 and A3 B-2 with TEG-Ts to give, respectively, ABAB-1 and A3 B-1 took place in poor yields (15% and 13%, respectively), making this linear approach less efficient than the preparation of A-1 and its condensation with B. It is worth mentioning that the corresponding symmetric A4 Zn(II)Pc is not detected quantitatively in any reaction mixture. This symmetric octasubstituted, TEG-Zn(II)Pc A4 -1 is useful to perform a full comparative study of the influence of the bulky fluorinated substituents and the TEG moieties on the solubility, aggregation and 1 O2 generation studies of this family of Zn(II)Pcs. In this context, this molecule was synthesized through a direct self-condensation of A-1 phthalonitrile, using in this case the classical pentanol/DBU conditions, which gave rise to A4 -1 in 11% yield. Figure 1 shows the 1 H NMR spectra of ABAB-1, A3 B-1, and A4 -1. The signal pattern for each compound arises from the molecular symmetry group these compounds belong to, namely, D2h for ABAB-1, C2h for A3 B-1 and D4h for A4 -1. Thus, highly symmetrical A4 -1 presents less signals than ABAB-1, which at the same time exhibits a simpler spectrum than A3 B-1. The 1 H NMR of ABAB-1, shows four singlets for the aromatic protons of the isoindole units and the trifluoromethylphenyl groups, two doublets for the phenoxy substituents, and a set of signals for the TEG chains with a chemical shift between 3.0 and 4.5 ppm. A3 B-1 produces six singlets for the isoindole units and the trifluoromethylphenyl substituents, and a multiplet that corresponds to the presence of three types of phenoxy groups. Figure 1 also shows structural models for the three compounds, where the molecular amphiphilic nature is represented in blue (hydrophilic zone) and yellow (hydrophobic zone). The products show good solubility in polar media, and, although they are no soluble directly in water due to the strong amphiphilic character, they are soluble in DMSO/water or MeOH/water (1:99) mixtures. Molecules 2020, 25, 213 4 of 18 Molecules 2019, 24, x FOR PEER REVIEW 4 of 17 Figure 1. 1.1 H1HNMR Figure NMRspectra spectrafor forABAB-1 ABAB-1 (in (in CDCl CDCl33), A33B-1 B-1 (in (in THF-d THF-d8)8 )and andAA 4-1 4 -1(in (inDMSO-d DMSO-d6). 6 ). Geometry Geometry optimization optimization (MM2,SCIGRESS (MM2, SCIGRESS(FJ (FJ2.8.1 2.8.1EU EU 3.3.1)) 3.3.1)) for each each molecule moleculeisisshown. shown. 2.2.2.2. Photophysical Characterization Photophysical Characterization The UV–Vis The UV–Vis spectra spectra ofofABAB-1 ABAB-1and 3B-1, in andAA3 B-1, in both both non-coordinating non-coordinating toluenetolueneand andcoordinating coordinating THF, THF,showshow splitQ-bands split Q-bandsaccompanied accompanied by the the typical typicalvibrational vibrational absorptions, absorptions, although although splitting splitting is much more pronounced is much pronouncedininABAB-1, ABAB-1,which which is is consistent consistent with its its with D2hD symmetry 2h symmetry (Figures S1 and (Figures S1 S2 and S2 and and Table Table 1). 1). Indeed, Indeed,the theabsorption absorptionspectraspectraofofthesethesecompounds compoundsdodonot notshow showany anyevidence evidence of of aggregation, aggregation, as otherwise as otherwise expected expected due due to thetopresence the presence of rather of rather bulky substituents bulky substituents at the non- at the non-peripheral peripheral positions of the Pc core. Further confirmation of the lack positions of the Pc core. Further confirmation of the lack of aggregation for these Zn(II)Pc of aggregation for these Zn(II)Pc results results from absorption studies performed at a range of concentrations from absorption studies performed at a range of concentrations (between ~0.5·10 M – 5·10−6 M) (between ∼0.5·10 −6 −6 M – 5·10 −6 M) (Figures (Figures S1 andS1S2),andwith S2), an with an analysis analysis of linear of linear regression regression between between thethe intensity intensity of of thethe Q-band Q-band andthe and the concentration giving R 2 values of ∼0.999. A4-1, with a regular single Q-band in its UV–Vis 2 concentration giving R values of ~0.999. A4 -1, with a regular single Q-band in its UV–Vis spectrum, spectrum, does not show either any evidence of aggregation in these solvents, probably because the does not show either any evidence of aggregation in these solvents, probably because the phenyloxy phenyloxy substituents hamper π–π stacking of the Pc cores as well (Figures S1 and S2). Increasing substituents hamper π–π stacking of the Pc cores as well (Figures S1 and S2). Increasing the number of the number of α-bis(trifluoromethyl)phenyl substituents produces a red-shift of the Q-band α-bis(trifluoromethyl)phenyl substituents produces a red-shift of the Q-band maximum, namely, from maximum, namely, from 682 nm for A4-1 to 712 nm for ABAB-1 in toluene solutions (Table 1). On 682 nm for A4 -1 to 712 nm for ABAB-1 in toluene solutions (Table 1). On the other hand, fluorescence the other hand, fluorescence studies have been also performed in toluene and THF solutions of studies have been also performed in toluene and THF solutions of ABAB-1, A B-1 and A4 -1, showing ABAB-1, A3B-1 and A4-1, showing an increase in the Stokes shift 3 as the number of an bis(trifluoromethyl)phenyl increase in the Stokes shift asunits the number attached of bis(trifluoromethyl)phenyl to the Zn(II)Pc increases units (Figure attached to the Zn(II)Pc S3). Fluorescence increases quantum (Figure yield S3). Fluorescence (ΦF) (Figure S4), and quantum singlet (yield τS) and(Φtriplet F ) (Figure state S4), and singlet (τT) lifetimes (τS ) andfrom (extracted triplet state Time- (τTResolved ) lifetimesFluorescence (extracted from Time-Resolved Fluorescence -TRF- experiments in -TRF- experiments in Figure S5) are included in Table 1. The results are Figure S5) are included in Table 1. The consistent withresults those are consistent withZn(II)Pcs, of non-aggregated those of non-aggregated and show a modest Zn(II)Pcs, solventand show a modest dependence. The solvent dependence. The mono-exponential mono-exponential fluorescence fluorescence decay kinetics confirmdecaythat kinetics ABAB-1, confirm A3B-1,that andABAB-1, A4-1 areA3in B-1, andmonomeric A4 -1 are informmonomeric in THF and form in THF toluene and toluene solutions, solutions, and the and the singlet singlet excited-state excited-state lifetimes are similarlifetimes for arethe similar for the two solvents. two solvents. The The quantification of quantification the φφ of the Δ, ∆for ABAB-1, , for ABAB-1, A3B-1,A3and B-1,Aand4-1 has A4been -1 hasperformed by two different been performed by two methods. different The firstThe methods. onefirst entails one the measurement entails the measurementof the photoinduced decomposition of the photoinduced of 1,3- decomposition Molecules 2020, 25, 213 5 of 18 of 1,3-diphenylisobenzofuran (DPBF) in DMSO solutions, under the irradiation of the corresponding Zn(II)Pcs. The spectroscopic data obtained from these measurements are shown in Figure S6. For the three compounds, the decrease in the DPBF absorption was completely linear (R2 = 0.99), while no change was observed in the absorption of Zn(II)Pcs during the measurement, which evidences that photobleaching is not taking place under the irradiation conditions. ABAB-1, A3 B-1, and A4 -1 showed Φ∆ values of 93%, 85%, and 53%, respectively. These values should be considered with caution since indirect methods could be subjected to different variables, but are useful for comparative purposes. From those results we can infer that the larger the number of bulky fluorinated groups at α-positions, the better the 1 O2 generation efficiency, as otherwise expected considering previous studies with related compounds [22], which show the important role of the bulky, electron-withdrawing bis(trifluoromethyl) groups in the 1 O2 production. It is important to mention that aggregation is not considered to evaluate these results since the three compounds are non-aggregated in DMSO (see below). On the other hand, we have evaluated the Φ∆ for ABAB-1, A3 B-1, and A4 -1 via the direct detection of 1 O2 phosphorescence at 1275 nm exciting at 355 nm, both in toluene and THF solutions (Figure S7 and Table 1). The Φ∆ values show the same trend observed in the indirect measurements, namely, a parallel increase with the number of bulky fluorinated substituents and, indeed, ΦF and Φ∆ change concomitantly in the opposite direction. Overall, the measured Φ∆ values are moderately higher than those reported for other amphiphilic Zn(II)Pcs, which confirms that the substitution pattern of this family of compounds improves the photophysical properties of the dye core, rendering them adequate for PDT applications. Importantly, we tried to perform measurements of 1 O2 generation in water, but only residual signals were detected, which can be rationalized by the formation of aggregates (see below). Table 1. Photophysical properties of Zn(II)Pcs ABAB-1, A3 B-1 and A4 -1 in toluene and THF. Sample Solvent Logε (λ/nm) λf /nm ΦF τS /ns τT /µs Φ∆ ABAB-1 toluene 4.73 (328), 5.09 (674), 5.12 (712) a 715 0.13 1.7 b 0.77 0.35 A3 B-1 toluene 4.63 (349), 4.97 (679) a , 4.94 (697) 702 0.17 2.1 0.47 b 0.71 A4 -1 toluene 4.50 (349), 5.03 (682) a 684 0.43 3.0 0.45 b 0.61 ABAB-1 THF 4.82 (349), 5.08 (671), 5.09 (703) a 709 0.13 1.7 0.30 c 0.83 A3 B-1 THF 4.71 (348), 5.03 (675) a 692 0.28 2.2 0.23 c 0.74 A4 -1 THF 4.61 (349), 5.12 (676) a 682 0.25 3.0 0.37 c 0.68 a λ absorption maxima; b uncertainty of ± 0.05; c uncertainty of ±0.08. 2.3. Aggregation Experiments Pcs have tendency to form aggregates from their aromatic systems for minimizing the solvation energy, particularly in the case of polar solvents as water. Although a certain organization of the dyes in vesicles or micelles can facilitate their transport through the blood system, it is important that once the PS is delivered to the target tissue, it remains as non-aggregated species, at least in some extent, that leading to more efficient 1 O2 generation through their photoexcitation. For that reason, aggregation studies are important for the former evaluation of their potential application in vitro. Absorption and emission experiments were carried out over solutions of TEG-functionalized ABAB-1, A3 B-1, and A4 -1. Although they are not soluble in pure water, they appear to be soluble upon addition from stock DMSO solutions to Milli-Q water until a 1:99 DMSO/water ratio is reached, what is admissible for further in vitro assays. Therefore, we performed aggregation studies by registering UV–Vis and fluorescence spectra of the three Zn(II)Pcs in different mixtures of DMSO/water, in order to compare the specific behavior derived from the presence of two substituted isoindole units with bulky fluorinated groups (ABAB-1) versus one (A3 B-1) or none of them (A4 -1). Mixed solutions of DMSO/water ranging from 100:0 to 1:99 were measured, in both absorption (Figure 2) and emission (Figure S8) experiments. Importantly, initial measurements in pure DMSO correspond to a fully non-aggregated state for the three compounds. Looking at the absorbance spectra, we can determine that from 100% to 80% DMSO, ABAB-1 presents an almost constant absorption for the Q-band, whereas A3 B-1 shows a gradual Molecules 2019, 24, x FOR PEER REVIEW 6 of 17 Molecules 2020, 25, 213 6 of 18 Molecules 2019, 24, x FOR PEER REVIEW 6 of 17 wavelength, compatible with the formation of co-facial H-type aggregated species. Furthermore, in wavelength, the case of decrease compatible ABAB-1, of its intensity when andwith a the formation 70:30 broadening, DMSO/water as wellof asco-facial aratio H-type rise ofis bandaggregated a reached, withthe species. spectrum shorter Furthermore, shows wavelength, a sudden compatible in the decreasecase of ABAB-1, of the Q-band with the formation when a 70:30 intensity, of co-facial H-typeand DMSO/water the concomitant aggregated ratio is reached, the emergence inofthe species. Furthermore, spectrum a case shoulder shows a sudden of at when of ABAB-1, lowera decrease wavelength. of the Below Q-band 70% of intensity, DMSO and contents, the we concomitant can observe a emergence constant 70:30 DMSO/water ratio is reached, the spectrum shows a sudden decrease of the Q-band intensity, of behavior a shoulder for both of ABAB-1at lower and and wavelength. Athe 3B-1 compounds, concomitant Below 70% of indicating emergence DMSO of that contents, a stable a shoulder we ofsituationcan observe a constant is reached. InBelow at lower wavelength. behavior the case 70%ofofADMSO for both 4-1, a sudden ABAB-1 contents, weand decrease can A of B-1 Q-band, 3the observe compounds, a constantas wellindicating forthat as a broadening behavior a stable both andsituation ABAB-1 and Aof3isB-1 rising areached. band at In compounds, the indicating shorter case of A4-1, wavelength thata compatible sudden a stable decreasewith situation of the the Q-band, formation In of as wellofasAa -1, aggregates, broadening and rising atof aQ-band, band at as shorter wavelength compatible with is reached. the case 4 area observed starting sudden decrease of 80:20 the DMSO/water well asratios. In the case a broadening of rising and emission of a the formation studies, band at we of aggregates, can wavelength shorter are discern thatcompatible observed the fluorescence starting with the at 80:20 of formation DMSO/water A3B-1 andofAaggregates, 4-1 shows aare ratios. sharp In the case decrease observed of emission in intensity starting at 80:20 studies, when thewe DMSO/water can water discern proportion ratios. that In the theoffluorescence increases, case of Acancellation until astudies, emission total 3B-1 and A4-1 we can discern ofshows a sharp fluorescence that the atdecrease ofinAintensity 70:30 DMSO/water fluorescence 3 B-1 and when ratio. the A4 -1 shows water On thea other proportion sharp hand, decrease increases, ABAB-1 until shows in intensity when a total a similar cancellation the water behavior of fluorescence than increases, proportion that observed at 70:30 until in DMSO/water the cancellation a total absorption ratio. spectrum, On that of fluorescencethe other is, hand, ata70:30 huge ABAB-1 decrease DMSO/water of shows the a similar emission ratio. On theafter behavior other the thanofthat addition hand, ABAB-1 30% observed showswater. Ain a similar the absorption magnification behavior of than spectrum, that observed in the absorption spectrum, that is, a huge decrease of the emission after the addition of this spectrum that is, shows a huge us decrease that since of thisthe emission moment a after residualthe addition fluorescence of 30% water. signal is A kept magnification with the same of this 30% spectrum intensity, water.which shows meansusthat A magnification thatwe since of have this this moment reach spectrum a residual a balanced shows that fluorescence ussituationsincewith thisthe signal presence moment aisresidual kept with the same of non-aggregated fluorescence intensity, species signal is which inkept the means medium. with that we the same have reach intensity, which a balanced means that situation we have with theapresence reach balancedof non-aggregated situation with the species in the medium. presence of non-aggregated species in the medium. ABAB 1 in DMSO:water DMSO A3B 1 in DMSO:water A4 1 in DMSO:water DMSO DMSO 0.4 0.4 ABAB 1 in DMSO:water 100% DMSO A3B 1 in DMSO:water 100% 0.4 A4 1 in DMSO:water DMSO 100% DMSO 0.4 90% 0.4 0.4 100% 90% 0.3 100% 90% 100% / a.u./ a.u. 0.3 / a.u./ a.u. 80% 90% 0.3 / a.u./ a.u. 80% 90% 80% 90% 0.3 0.3 0.3 70% 80% 70% Absorbance 80% 70% Absorbance 0.2 80% Absorbance 0.2 0.2 50% 70% 50% 70% 50% Absorbance 70% Absorbance 0.2 Absorbance 0.2 0.2 30% 50% 30% 50% 30% 0.1 0.1 50% 0.1 10% 30% 10% 30% 10% 30% 0.1 0.1 0.1 1% 10% 1% 10% 1% 10% 0 0 0 300 400 500 600 700 800 1% 300 400 500 600 700 800 1% 300 400 500 600 700 800 1% 0 0 0 300 400 500 600 Wavelength / nm 700 800 300 400 500 600 Wavelength / nm 700 800 Wavelength 300 400 500 600 / nm 700 800 Wavelength / nm Wavelength / nm Wavelength / nm Figure Figure2.2.UV–Vis UV–Visspectra spectraof ofABAB-1, ABAB-1,A A33B-1, B-1,and andAA4-1 in different DMSO/water ratios. 4 -1 in different DMSO/water ratios. Figure 2. UV–Vis spectra of ABAB-1, A3B-1, and A4-1 in different DMSO/water ratios. 2.4. 2.4.Determination Determinationofofthe theLog LogPo/w Po/w 2.4. Determination of the Log Po/w For Foraapreliminary preliminaryevaluation evaluationof ofthe the relative relative lipophilicity lipophilicity ofof ABAB-1, ABAB-1, A A33B-1, B-1,andandA A44-1, -1,andandtheirtheir For affinity affinityfora preliminary for cell membranes, evaluation membranes, n-octanol/PBS of the relative n-octanol/PBSpartition lipophilicity partitioncoefficients of coefficients ABAB-1, were were A determined determined 3 B-1, and using A using -1, the the 4 and their shake- shake-flask affinity flask method for method (seecell (seemembranes, Figure Figure S9),S9), whichn-octanol/PBS which doesdoesnotnot partition require require coefficients standard standard were determined compounds compounds andisis based and based usingonthe theshake- the direct direct flask method determination determinationof (see Figure ofequilibriumS9), which equilibriumpartition does not require partitionconcentrations concentrations of standard compounds ofaacompound compound in and inaabiphasic is based biphasicsystem. on system.Althoughthe direct Although determination the the three compounds of equilibrium compounds have have apartition a commoncommon concentrations structural structural Pcofthey Pc core, a compound core, in a biphasic they exhibited exhibited different system. different amphiphilic Although amphiphilic character. the threerenders character. ABAB-1 compounds ABAB-1 higherhave a renders a ahigher common octanol/water structural octanol/water Pc core, coefficient partition partition coefficient they than exhibited A3 B-1thananddifferent AA3B-14 -1, and (seeamphiphilic A 4-1, (see Figure 3), character. Figure for [Zn(II)Pc]ABAB-1 −5 3), for [Zn(II)Pc] renders = 10 M =in10theM a −5 higher octanol/water in the octanol/water octanol/water partition mixture mixture (%DMSO coefficient (%DMSO than < 1%), thus < 1%), A B-1 and thus indicating 3 indicating A 4 -1, the relative (see the Figure order of3), relative for of order [Zn(II)Pc] lipophilicity = 10−5nature. lipophilicity nature. InMfact, in the In octanol/water thefact, log Pthe/ O Wlog P values O /Wmixture values suggest(%DMSO suggest that < that ABAB-1 1%), ABAB-1 and thus A 3 indicating and B-1 A are3B-1 the are mostly relative mostly orderand hydrophobic of lipophilicity hydrophobic have and have very highnature. very In fact, high affinity the log affinity for forPmembranes. membranes. O/W values suggest thethat Onhand, On the other A4ABAB-1 other -1 hand, is more and 4-1Ais 3B-1 Aamphiphilic, moreare mostly hydrophobic amphiphilic, and its presenceandinitsthe and have presence very aqueousinphase high the aqueous affinity for phase is more is more abundant, membranes. abundant, which On the other whichwhich is concordant hand, is concordant A its higherwhich 4 -1 is more solubilityits amphiphilic, higher andinitsthis in this solubility medium. presence medium. in the aqueous phase is more abundant, which is concordant which its higher solubility in this medium. Figure Figure 3. 3. n-octanol/water n-octanol/waterpartition partitionexperiments experiments with with ABAB-1, ABAB-1, A A33B-1, B-1,and andAA4-1. 4 -1. Figure 3. n-octanol/water partition experiments with ABAB-1, A3B-1, and A4-1. Molecules 2020, 25, 213 7 of 18 2.5. Biological Studies Biological experiments with human HeLa (cervical adenocarcinoma cells) and SCC-13 (skin squamous carcinoma cells) strains have been performed using ABAB-1, A3 B-1, and A4 -1 as PS, in order to evaluate the potential of our design. 2.5.1. Cytotoxicity Studies Phototoxicity of some Zn(II)Pcs has already been tested against cervical and oral skin squamous carcinoma cells in in vitro experiments. However, due to the aggregation problems they present, it is necessary to transport them through complex conjugates [26–28]. Herein, we have studied the activity of ABAB-1, A3 B-1 and A4 -1 Zn(II)Pcs as independent molecular entities upon red light irradiation by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in SCC-13 and HeLa cell lines. For this purpose, we first evaluated the inherent toxicity of two different concentrations of the three Zn(II)Pcs (1·10−6 and 1·10−7 M) in cells, after 5 h of incubation in presence of PS in the dark. Separately, we have evaluated the possible damage caused by irradiation, treating the cells with the highest red light dose used in this work (9 J/cm2 ), in the absence of the PS. The results shown in Table 2 indicate that neither the presence of the Zn(II)Pc at these concentrations, nor the administration of red light, induced significant cytotoxic effects in the cell lines studied, and survival rates above 95% were obtained. In addition, we have analyzed whether the percentage of DMSO influences the single action of the PS or light, not observing changes in cell survival when combined with DMSO (Table 2). Table 2. Toxicity effects in SCC-13 and HeLa cells induced by red light, in the absence of any Pcs, and by incubation with either of the Zn(II)Pcs 1 in the dark. Cell toxicity was evaluated by the MTT assay, 24 h after treatment. Surviving Fraction (% ± SD) 1 Compound Concentration [M] SCC-13 HeLa No Zn(II)Pc – Control dark 101 ± 2.4 100 ± 1.1 No Zn(II)Pc – Control dark + 1% DMSO 102 ± 4.3 98 ± 3.7 No Zn(II)Pc – Control light (9 J/cm2 ) 100 ± 1.7 99 ± 2.3 No Zn(II)Pc – Control light (9 J/cm2 ) + 1% DMSO 101 ± 2.8 101 ± 1.5 1 × 10−6 98 ± 5.4 101 ± 5.5 ABAB-1 1 × 10−7 102 ± 3.1 95 ± 2.8 1 × 10−6 97 ± 6.3 97 ± 3.5 A3 B-1 1 × 10−7 99 ± 5.5 99 ± 3.6 1 × 10−6 99 ± 4.5 97 ± 5.5 A4 -1 1 × 10−7 98 ± 5.0 98 ± 2.8 1 Data are expressed as mean values obtained from three independent experiments ± standard deviation (SD). Then, we evaluated the photodynamic activity of ABAB-1, A3 B-1, and A4 -1 toward SCC-13 and HeLa cells upon irradiation. For the photodynamic treatments, we used two different concentrations (i.e., 1·10−6 and 1·10−7 M) and, after 5 h of incubation, cells were exposed to different red light doses (3, 6 and 9 J/cm2 ). It is important to mention here that, for a better comparison between the three compounds, one should calculate the total number of photons absorbed by each. However such calculation is very challenging, if not impossible, because they are likely to be aggregated to different extents, which leads to severe spectral changes (see Figure 2). Moreover, their distribution in the different cell compartments may be different, adding further uncertainty to the absorbance of the active species responsible for cell death. Thus, the only meaningful comparison is by concentration. Figure S10 shows concentration-dependent response for the three compounds; cells were more sensitive to PDT when higher concentration (1·10−6 M) were applied. We selected the concentration of 1·10−6 M to perform the rest of experiments. Figure 4 shows the results for both cell lines treated with the Zn(II)Pcs; a drastic decrease in cell survival was revealed with the light dose in both cells lines. ABAB-1 was the most sensitive at low red light dose, but the results for ABAB-1 and A3 B-1 Molecules 2020, 25, 213 8 of 18 Molecules 2019, 24, x FOR PEER REVIEW 8 of 17 were similar when using 6 or 9 J/cm2 irradiations. Notably, the measured cell survival values are lines. actually ABAB-1 was comparable to the thosemost sensitive at low of monomeric red light dose, Si(IV)Pc-PEG but the species, results which for ABAB-1 present and Afor IC50 values 3B-1HeLa were similar when using 6 or 9 J/cm 2 irradiations. Notably, the measured cell survival values are cells of 0.28 µM [20], and also to other tetra-PEG functionalized Zn(II)Pcs [29]. On the other hand, actually comparable to those of monomeric Si(IV)Pc-PEG species, which present IC50 values for HeLa A4 -1 was always less efficient in comparison with the other Zn(II)Pcs, leading to only 20% of cell cells of 0.28 μM [20], and also to other tetra-PEG functionalized Zn(II)Pcs [29]. On the other hand, A4- death in both cell lines with the highest light dose (i.e., 9 J/cm2 ) (Figure 4a). Importantly, the trend 1 was always less efficient in comparison with the other Zn(II)Pcs, leading to only 20% of1cell death foundinfor both cell survival cell lines withvalues is parallel the highest light to dosethe(i.e. relative 9 J/cmefficiency of the 2) (Figure 4a). three Pc dyes Importantly, as O the trend 2 generators found for (Tablecell 1).survival Cell morphology of the treated cells was also analyzed 24 h after PDT using values is parallel to the relative efficiency of the three Pc dyes as O2 generators (Table 1 phase contrast microscopy 1). Cell (Figure morphology 4b), revealing cytoplasmic of the treated cells wasretraction, with24 also analyzed a rounded h after PDTaspectusingsimilar phasetocontrast that of cells in apoptosis, microscopy in both cell4b), (Figure lines treated cytoplasmic revealing with ABAB-1 and A3 with retraction, B-1 ata higher rounded light aspect doses [30].toInterestingly, similar that of thesecells imagesin apoptosis, in both cell are well correlated lines with thetreated resultswith ABAB-1 obtained fromand cellA3viability B-1 at higher assays. light Wedoses next [30]. analyzed Interestingly, these images are well correlated with the results obtained from by fluorescence microscopy the intracellular ROS formation in SCC-13 and HeLa cells when subjected cell viability assays. We next analyzed by fluorescence microscopy the intracellular ROS formation to PDT with Zn(II)Pcs ABAB-1, A3 B-1 and A4 -1, using for this purpose the DHF-DA fluorescent probe in SCC-13 and HeLa cells when subjected to PDT with Zn(II)Pcs ABAB-1, A3B-1 and A4-1, using for this purpose the DHF-DA (Figure S11, Supporting Information). As shown in Figure S11, controls and cells incubated with fluorescent probe (Figure S11, Supporting Information). As shown in Figure S11, controls and cells Zn(II)Pcs in dark conditions did not exhibit green florescent due. In contrast, cells subjected to PDT incubated with Zn(II)Pcs in dark conditions did not exhibit green florescent due. In contrast, cells showed an intense subjected to PDT fluorescence in bothfluorescence showed an intense cell types with thecell in both twotypes of the compounds, with the two of the ABAB-1 and A3 B-1 compounds, (compared ABAB-1 toand baseline A3B-1levels) revealing (compared a prominent to baseline ROS production levels) revealing a prominent after ROSPDT. However, production cells after treated PDT. with However, A4 -1 presented a very cells treated low with fluorescence, A4-1 presented a very indicating a low ROS low fluorescence, production indicating a lowthatROSis in agreement production with that the cell is insurvival agreement results. with the cell survival results. Figure Figure 4. (a)4.Phototoxicity (a) Phototoxicity induced induced bybyABAB-1, ABAB-1, A A33B-1, B-1,and andAA 4-1 in SCC-13 and HeLa cells. Cells were 4 -1 in SCC-13 and HeLa cells. Cells were incubated with concentrations of 1·10 −6 −6 for 5 h and then irradiated with red light at variable doses. incubated with concentrations of 1·10 for 5 h and then irradiated with red light at variable doses. The The survival was dependent of red light dose; A4-1 was the less sensible to light irradiation. Cell survival was dependent of red light dose; A4 -1 was the less sensible to light irradiation. Cell survival survival was evaluated by the MTT test 24 h after treatments. Each value corresponds to the mean was evaluated by the MTT test 24 h after treatments. Each value corresponds to the mean obtained obtained from three independent experiments ± SD. (* p < 0.05; ** p < 0.01; *** p < 0.001). (b) from three independent experiments ± SD. (* p < 0.05; ** p < 0.01; *** p < 0.001). (b) Morphological Morphological changes observed in SCC-13 and HeLa cells 24 h after photodynamic treatment with changes observed the three in −6SCC-13 Pcs (1·10 and HeLafollowed M, 5 h incubation cells 24byh 9after J/cmphotodynamic 2 of red light). treatment with the three Pcs −6 2 (1·10 M, 5 h incubation followed by 9 J/cm of red light). Molecules 2020, 25, 213 9 of 18 Molecules 2019, 24, x FOR PEER REVIEW 9 of 17 2.5.2. 2.5.2. Localization Localization Study Study To To localize the Zn(II)Pcs Zn(II)Pcs in in the the treated treatedcells, cells,they theywere wereirradiated irradiatedwithwithgreen greenlight light(545 (545nm) nm)with witha aBP545 BP545filter. filter.InInFigure Figure55we wecan cansee seethat thatABAB-1, ABAB-1,AA33B-1,B-1,and andA A44-1 -1 are are located located inside the cells. They They appear with aavesicular appear with vesicularmorphology morphology and and they they areare probably probably localized localized in lysosomes in lysosomes or endosomes, or endosomes, which which suggestsuggest that theythat they are internalized are internalized through anthrough endocyticanpathway. endocytic pathway. Unfortunately, Unfortunately, the fluorescencethe of fluorescence these Zn(II)Pcs of these is tooZn(II)Pcs weak to isbetoo weak to beifappreciated appreciated if specific specific probes for eachprobes for each organelle areorganelle are used, which used, which fluorescence fluorescence mask the mask the emission emission of the of the Zn(II)Pcs. Zn(II)Pcs. For the sake For the sake of comparison, of comparison, we are we are including in including AppendixinAAppendix (Figure A1) A images (Figure of A1)subcellular images of localization subcellular localization of organelles of for organelles SCC-13 for andSCC-13 and HeLa cells HeLa cells after incubation after incubation with known with known fluorescent fluorescent probes. probes. Figure 5. Figure Cellular localization 5. Cellular localization ofof ABAB-1, ABAB-1, AA33B-1, andA B-1,and (red)in A44-1-1(red) inSCC-13 SCC-13and andHeLa HeLacells cellswhen when they they are observed are observed with with aa fluorescence fluorescence microscopy microscopy (irradiated with green light at 545 545 nm) nm) and and without without other other cell dye. cell dye. They Theyappear appear withwith aa vesicular vesicular morphology morphology inside inside cells. cells. 3. Materials and Methods 3. Materials and Methods 3.1. General Methods and Characterization Techniques 3.1. General Methods and Characterization Techniques Chemical reagents were purchased from Merck-Sigma Aldrich, Alfa Aesar, Acros Organics, TCI Chemical reagents or Fluorochem, as mainwere purchased commercial from Merck-Sigma suppliers, Aldrich, and were used Alfa Aesar, without furtherAcros Organics, purification TCI unless or Fluorochem, as main commercial suppliers, and were used without further purification it is indicated. Solvents were purchased from Carlo Erba Reagents, and anhydrous solvents were unless it is indicated. dried Solvents with 4Å were sieves molecular purchased from Carlo (Panreac). Erba Reagents, All reactions were and anhydrous performed solvents glasswares, in standard were dried with 4Å molecular sieves (Panreac). All reactions were performed in standard glasswares, except for the cyclotetramerization reactions that were carried out in high pressure sealed tubes. exceptThe for the cyclotetramerization monitoring reactions of the reactions that were has been carried carried outthin out by in high layerpressure sealed tubes. chromatography The monitoring (TLC), employing of the reactions has been carried out by thin layer chromatography (TLC), aluminum sheets coated with silica gel type 60 F254 (0.2 mm thick, E. Merck). Purificationemploying aluminum and sheets coated with silica gel type 60 F254 (0.2 mm thick, E. Merck). Purification separation of the synthesized products was performed by column chromatography, using silicaand separation of the gel synthesized products was performed by column chromatography, using silica (230–400 mesh, 0.040–0.063 mm, Merck) and eluents are indicated for each particular case. Gelgel (230–400 mesh, 0.040-0.063 permeation mm, Merck) and(GPC) chromatography eluents wasare indicated performed for Bio-Beads using each particular case. Gel S-X1 (200–400 mesh,permeation Bio-Rad). chromatography (GPC) was performed using Bio-Beads S-X1 (200–400 mesh, Bio-Rad). Infrared (IR) spectra were recorded on a Cry 630 FTIR spectrophotometer from Agilent Technologies, Infrared (IR) spectra were using solid recorded samples on a Cry (diamond 630 FTIR ATR). spectrophotometer High-resolution from Agilent mass spectrometry Technologies, (HR-MS) using using were recorded solid samples (diamond ATR). High-resolution mass spectrometry (HR-MS) were recorded positive ESI Positive TOF_MS or matrix-assisted laser desorption/ionization (MALDI). Electrospray using positive ESI Positive TOF_MS or matrix-assisted laser desorption/ionization (MALDI). Electrospray ionization (ESI) mass spectra were recorded with an API Q-Star Pulsar i from Sciex. Matrix used are ionization (ESI) mass spectra were recorded with an API Q-Star Pulsar i from Sciex. Matrix used are indicated in each spectrum. MALDI-TOF HR-MS spectra were recorded with a Bruker Ultrareflex indicated in each spectrum. MALDI-TOF HR-MS spectra were recorded with a Bruker Ultrareflex III III spectrometer. MS data are expressed in m/z units. All MS experiments were carried out at the spectrometer. MS data are expressed in m/z units. All MS experiments were carried out at the Servicio Servicio Interdepartmental de Investigación (SIdI) of the Universidad Autónoma de Madrid. Nuclear Interdepartmental de Investigación (SIdI) of the Universidad Autónoma de Madrid. Nuclear magnetic resonance spectra (1H NMR and 13C NMR) were recorded on Bruker AC-300 (300 MHz) or Molecules 2020, 25, 213 10 of 18 magnetic resonance spectra (1 H NMR and 13 C NMR) were recorded on Bruker AC-300 (300 MHz) or Bruker XRD-500 (500 MHz) instruments. Deuterated solvents employed are indicated in each spectrum. Aggregation studies were performed using aqueous miliQ-water, and organic spectroscopy grade solvents. UV–Vis spectra for aggregation experiments were recorded with a UV-vis JASCO-V660 spectrophotometer using 10 × 10 mm quartz cuvettes. 3.2. Synthesis Compounds 3,3”,5,5”-tetrakis(trifluoromethyl)-[1,10:4’,1”-terphenyl]-20,30-dicarbonitrile (B) [11], 4,5-bis(4- hidroxiphenoxy)phthalonitrile (A-2) [23], 2-(2-(2-methoxyethoxy)ethoxy)ethyl p-toluenesulfonate (TEG-Ts) [25], and ABAB Zn(II)Pc 2 [24], have been prepared according to published procedures. 4,5-bis-(4-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)phenoxy)phthalonitrile (A-1): In a two-neck round bottom flask, A-2 (150 mg, 0.44 mmol) and TEG-Ts (291 mg, 0.91 mmol) were solved in dry DMF (6 mL) and heated to 50 ◦ C. Then, anhydrous K2 CO3 (199 mg, 1.44 mmol) was added in several portions. Reaction was kept at this temperature under argon atmosphere for three days. After cooling the reaction mixture was diluted with 100 mL of water and extracted with EtOAc (3 × 50 mL). The collected organic phase was washed with water (3 × 50 mL), brine (50 mL) and dried with MgSO4 . The solvent was filtered and evaporated under reduced pressure. Then, product was purified by column chromatography on SiO2 with EtOAc as eluent, resulting in 275 mg (98% yield) of a brown oil. 1 H NMR (300 MHz, CDCl3 ): δ 3.38 (s, 6H, TEG-CH3 ), 3.53–3.62 (m, 4H, TEG-CH2 ), 3.63–3.83 (m, 12H, TEG-CH2 ), 3.85–3.95 (m, 4H, TEG-CH2 ), 4.12–4.23 (m, 4H, TEG-CH2 ), 6.96–7.13 (m, 10H, CHAr). 13 C NMR (75 MHz, CDCl3 ): δ 59.2, 68.2, 69.8, 70.7, 70.8, 71.0, 72.1, 109.9, 115.3, 116.5, 120.7, 121.6, 147.4, 152.6, 157.0. IR (ATR) ν–1 (cm–1 ): 2875, 2229, 1499, 1292, 1246, 1206, 1105. HR-MS (ESI+, +TOF, Ionizing phase: MeOH + NaI) for C34 H40 N2 O10 : m/z 659.2568 [M + Na]+ , (calculated: 659.2575). Zn(II)Pc A3 B-2: Phthalonitrile B (150 mg, 0.27 mmol), phthalonitrile A-2 (93 mg, 0.27 mmol) and anhydrous Zn(AcO)2 (50 mg, 0.27 mmol) were placed in a 5 mL high pressure resistant flask equipped with a magnetic stirrer, and then 2.7 mL ([B] = 0.1 M) of dry o-DCB/DMF (2:1) were added. The mixture was heated to 150–160 ◦ C overnight under an argon atmosphere. After cooling the solvent was removed under vacuum. The product was purified by column chromatography on SiO2 (dioxane/heptane in gradient from 1:1 to 2:1) where the second fraction to elute containing the desired product A3 B-2. The product was further purified by an additional column chromatography on Bio-Beads using CHCl3 as eluent. After evaporation of the solvent blue solid was obtained, which were recrystallized from dichloromethane (DCM)/heptane. Yield: 16 mg (7%). 1 H NMR (300 MHz, DMSO-d6 ): δ 6.71–7.21 (m, 24H, O-Ph-O), 7.76 (s, 2H, CHAr), 8.04 (s, 2H, CHAr), 8.21 (s, 2H, CHAr), 8.63 (s, 4H, CHAr), 8.78 (s, 4H, CHAr), 9.28–9.56 (br s, 6H, OH). 13 C NMR (75 MHz, THF-d8 ): δ 116.1, 116.4, 116.5, 118.8, 120.1, 120.9, 121.7, 125.3, 129.4, 129.8, 130.2, 131.1, 131.7, 133.4, 133.6, 134.4, 135.8, 142.5, 148.9, 149.0, 149.7, 150.1, 150.7, 150.8, 151.2, 151.2, 151.3, 153.1, 153.2, 153.5, 153.6, 153.9, 154.6, 154.9. IR (ATR) ν–1 (cm–1 ): 3416, 2954, 2883, 1723, 1507, 1449, 1362, 1279, 1196, 1182, 1133, 1034. MS (MALDI, matrix DCTB + PEGNa 1500) for C84 H44 F12 N8 O12 Zn: m/z 1648.2177 [M]+ , (calculated: 1648.2173). Zn(II)Pc ABAB-1: Method a: Phthalonitrile B (150 mg, 0.27 mmol), and A-1 (172 mg, 0.27 mmol) and anhydrous Zn(AcO)2 (50 mg, 0.27 mmol) were placed in a 5 mL high pressure resistant flask equipped with a magnetic stirrer, and then 2.7 mL ([B] = 0.1 M) of dry o-DCB/DMF 2:1 were added. The mixture was heated to 150–160 ◦ C overnight under an argon atmosphere. After cooling the solvent was removed under vacuum. Then, the product was purified by column chromatography on SiO2 (heptane/dioxane 1:1 as eluent) where the first green fraction to elute contained the desired product. It was further purified by an additional column chromatography on Bio-Beads using CHCl3 as eluent. After evaporation of the solvent a blue solid was obtained, which was recrystallized from DCM/heptane. Yield: 33 mg (10%). Method b: ABAB-2 (10 mg, 0.0054 mmol) and TEG-Ts (34 mg, 0.11 mmol) were solved in dry DMF (7 mL) and heated to 50 ◦ C. Then, anhydrous K2 CO3 was added (7 mg, 0.048 mmol) and the reaction was kept under this temperature and argon atmosphere overnight. Molecules 2020, 25, 213 11 of 18 The reaction was monitored by TLC (heptane/dioxane) and extra anhydrous K2 CO3 additions could be needed until complete the conversion. After cooling the solvent was evaporated under reduced pressure. Then, the product was purified by column chromatography on SiO2 (heptane/dioxane 1:1 as eluent). It was further purified by an additional column chromatography on Bio-Beads using CHCl3 as eluent. After evaporation of the solvent a blue solid was obtained, which was washed with heptane. Yield: 2 mg (15%). 1 H NMR (500 MHz, CDCl3 ): δ 3.05 (s, 12H, TEG-CH3 ), 3.29 (m, 8H, TEG-CH2 ), 3.37 (m, 8H, TEG-CH2 ), 3.43 (m, 8H, TEG-CH2 ), 3.54 (m, 8H, TEG-CH2 ), 3.60 (m, 8H, TEG-CH2 ), 3.89 (m, 8H, TEG-CH2 ) 6.76 (d, 8H, J = 9.01 Hz, O-Ph-O), 6.98 (d, 8H, J = 9.01 Hz, O-Ph-O), 7.77 (s, 4H, CHAr), 7.85 (s, 4H, CHAr), 7.97 (s, 4H, CHAr), 8.52 (s, 8H, CHAr). 13 C NMR (125 MHz, CDCl3 ): δ 58.8, 67.6, 69.7, 70.2, 70.4, 70.7, 71.8, 114.5, 115.5, 118.7, 121.5, 123.6 (q, J = 271.6 Hz, CF3 ); 131.2, 131.5, 134.8, 135.3, 136.8, 142.6, 151.1, 151.9, 152.4, 153.4, 154.6. IR (ATR) ν–1 (cm–1 ): 2916, 2874, 1501, 1410, 1377, 1275, 1198, 1178, 1127, 1104, 896. HR-MS (MALDI, matrix: DCTB + PPGNa 2000) for C116 H96 F24 N8 O20 Zn: m/z 2440.5741 [M]+ , (calculated: 2440.5644). Zn(II)Pc A3 B-1: Method a: Phthalonitrile B (150 mg, 0.27 mmol), and A-1 (172 mg, 0.27 mmol) and anhydrous Zn(AcO)2 (50 mg, 0.27 mmol) were placed in a 5 mL high pressure resistant flask equipped with a magnetic stirrer, and then 2.7 mL ([B] = 0.1 M) of dry o-DCB/DMF 2:1 were added. The mixture was heated to 150–160 ◦ C overnight under an argon atmosphere. After cooling the solvent was removed under vacuum. Then, the product was purified by column chromatography on SiO2 (heptane/dioxane in gradient from 100:0 to 0:100) where the second green fraction to elute contained the desired product. It was further purified by an additional column chromatography on Bio-Beads using CHCl3 as eluent. After evaporation of the solvent a blue solid was obtained, which was recrystallized from DCM/heptane. Yield: 27 mg (8%). Method b: A3 B-2 (10 mg, 0.0061 mmol) and excess of TEG-Ts (39 mg, 0.12 mmol) were solved in dry DMF (7 mL) and heated to 50 ◦ C. Then, K2 CO3 (7.5 mg, 0.054 mmol) was added, and the reaction was kept under this temperature and argon atmosphere overnight. The reaction was monitored by TLC (heptane/dioxane) and extra anhydrous K2 CO3 additions could be needed until complete the conversion. After cooling the solvent was evaporated under reduced pressure. Then, the product was purified by column chromatography on SiO2 (heptane/dioxane in gradient from 100:0 to 0:100). It was further purified by an additional column chromatography on Bio-Beads using CHCl3 as eluent. After evaporation of the solvent a blue solid was obtained, which was recrystallized from EtOAc/heptane. Yield: 2 mg (13%). 1 H NMR (300 MHz, THF-d8 ): δ 3.27 (br s, 18H, TEG-CH3 ); 3.45 (br s, 12H, TEG-CH2 ), 3.52–3.75 (m, 36H, TEG-CH2 ); 3.85 (br s, 12H, TEG-CH2 ); 4.16 (br s, 12H, TEG-CH2 ); 6.93–7.18 (m, 24H, O-Ph-O); 7.98 (br s, 2H, CHAr); 8.10 (br s, 4H, CHAr); 8.74 (s, 4H, CHAr); 8.84 (s, 2H, CHAr); 8.87 (s, 2H, CHAr). 13 C NMR (75 MHz, THF-d8 ): δ 59.0, 69.0, 70.8, 71.5, 71.7, 71.8, 74.6, 116.0, 116.2, 116.6, 116.8, 119.1, 119.6, 120.3, 120.4, 122.2, 123.8, 131.7, 132.0, 132.2, 133.2, 135.6, 135.8, 135.9, 136.0, 136.1, 136.6, 137.5, 138.3, 152.3, 153.4, 156.0, 156.5. IR (ATR) ν−1 (cm−1 ): 2870, 1501, 1450, 1404, 1276, 1198, 1127, 1093, 1029, 890, 800. HR-MS (MALDI, matrix: DCTB + PPGNa 2000) for C126 H128 F12 N8 O30 Zn: m/z 2524.7894 [M]+ , (calculated: 2524.7831). Zn(II)Pc A4 -1: A mixture of A-1 (90 mg, 0.14 mmol) and Zn(OAc)2 (9.7 mg, 0.053 mmol) was placed in a 5 mL high pressure resistant flask equipped with a magnetic stirrer, and then 1.4 mL of pentanol and 0.1 mL of DBU were added. Then mixture was heated to 150 ◦ C under argon atmosphere for 1 h. After cooling the solvent was removed under vacuum. The product was purified by column chromatography on SiO2 (EtOAc/MeOH in gradient from 100:0 to 0:100). After evaporate the solvent under reduced pressure, the product was washed with DCM/heptane and a green solid was obtained. Yield: 9.7 mg (11%). 1 H NMR (500 MHz, DMSO-d6 ): δ 3.22 (s, 24H, TEG-CH3 ); 3.43 (t, 16H, J = 5.05 Hz, TEG-CH2 ); 3.54 (t,16H, J = 5.05 Hz, TEG-CH2 ); 3.58 (t, 16H, J = 4.94 Hz, TEG-CH2 ); 3.65 (t, 16H, J = 4.94 Hz, TEG-CH2 ); 3.82 (br t, 16H, TEG-CH2 ); 4.14 (br t, 16H, TEG-CH2 ); 7.04 (d, 16H, J = 8.70 Hz, O-Ph-O); 7.31 (d, 16H, J = 8.70 Hz, O-Ph-O); 8.40 (br s, 3H, CHAr-Pc(core)). 13 C NMR (125 MHz, DMSO-d6 ): δ 58.0, 67.6, 69.1, 69.6, 69.9, 70.0, 71.3, 112.3, 115.7, 119.8, 133.2, 149.8, 150.4, 154.9. IR (ATR) Molecules 2020, 25, 213 12 of 18 ν−1 (cm−1 ): 3016, 2933, 1502, 1450, 1425, 1400, 1284, 1200, 1130, 936. HR-MS (MALDI, matrix DCTB + PPGNa 2000 + PPGNa 2700) for C136 H160 N8 O40 Zn: m/z 2608.9966 [M]+ , (calculated: 2609.0018). 3.3. Spectroscopic Techniques Φ∆ in DMSO was measured following an indirect method based on the photoinduced decomposition of a scavenger, in this case DPBF which reacts with 1 O2 after the excitation with visible light filtered below 530 nm (hν > 530 nm) [31]. Non-substituted Zn(II)Pc was used as reference compound which present a Φ∆ = 0.67 in DMSO [32]. For these studies, a stock solution in DMSO of DPBF was prepared (with an absorbance of ~1 a.u.), and 2 mL were transferred into a 10 × 10 mm quartz optical cell and bubbled with 3 O2 for 1 min. Then, concentrated stock solutions of the corresponding Zn(II)Pcs in DMSO were added, in a defined amount to reach an absorbance of the final solution in the Q-band maximum of 0.1 ± 0.05 a.u. The solutions were stirred and irradiated for defined time intervals using a halogen lamp of 300 W. The length of time intervals was modified for each experiment, obtaining an absorbance diminution of DPBF ~3–4%. Incident light was filtered through a water filter (6 cm) and an additional filter to remove light under 530 nm (Newport FSQ-OG530 filter). Moreover, neutral density filters (FBS-ND03 and FB-ND10, were used). Decrease of DPBF absorbance with irradiation time was monitored at 414 nm and all experiments were performed three times. Average values are represented, and Φ∆ is calculated through Equation (1); R and S indicate reference and sample, respectively. k is the slope of a plot of ln (A0 /At ) versus irradiation time (t). A0 and At are the absorbance of DPBF at the monitored wavelength before and after irradiation time (t). IaT is the total amount of light absorbed by the dye. IaT is calculated as a sum of intensities of the absorbed light Ia at wavelengths from 530 nm to 800 nm. Ia at given wavelength is calculated using Beer’s law (Equation (2)), where I0 is the transmittance of the filter at a given wavelength and A is the absorbance of the dye at this wavelength. S R kS ·IaT R φ∆ = φ∆ · S (1) kR ·IaT   Ia = I0 · 1 − e−2.3A (2) All spectroscopic measurements for Photophysical Characterization (Section 2.2), were carried out in 1 cm quartz cuvettes (Hellma, Germany) in air-saturated solutions, at room temperature using spectroscopic grade solvents. Absorption spectra were recorded using a double beam UV–Vis–NIR Varian Cary 6000i spectrophotometer (Varian, Palo Alto, CA, USA). Absorption coefficients were derived from the slopes of Lambert-Beer plots. Fluorescence emission spectra were recorded using a Spex Fluoromax-4 spectrofluorometer (Horiba Jobin-Yvon, Edison, NJ, USA). The samples were excited at: ABAB-1 667 (THF) and 702 nm (toluene); A3 B-1 665 (THF) and 670 nm (toluene); A4 -1 693 (THF) and 672 nm (toluene). Fluorescent quantum yields (ΦF ) were determined by comparing the integrated fluorescent intensity of optically matched solutions between samples (S) and reference (R) ZnPc in 1-propanol (ΦF = 0.20) [22]. The samples were excited at: ABAB-1 609 (THF) and 603 nm (toluene); A3 B-1 610 (THF) and 605 nm (toluene); A4 -1 610.5 (THF) and 610 nm (toluene). Fluorescence intensity was corrected using the refractive index of the solvents used (Equation (3)). AreaS ·n2 S ΦF = (3) AreaR ·n2 R Time-resolved fluorescence decays were measured using a Fluotime 200 time-correlated fluorescence lifetime spectrophotometer (PicoQuant GmbH, Berlin, Germany), equipped with a red sensitive photomultiplier. Excitation was achieved by means of a 654 nm picosecond laser working at 1 MHz repetition rate. The counting frequency was always below 1%. Fluorescence lifetimes were analyzed using PicoQuant FluoFit 4.0 data analysis software. Molecules 2020, 25, 213 13 of 18 1O 2 generation in THF and toluene was studied by time-resolved near-infrared phosphorescence by means of a customized setup [33,34]. Briefly, a pulsed Nd:YAG laser (FTSS355-Q, Crystal Laser, Berlin, Germany) working at 1 kHz repetition rate (for THF and toluene) at 355 nm (third harmonic, 0.5 µJ per pulse) was used to excite the sample. A 1064-nm rugate notch filter (Edmund Optics) and an uncoated SKG-5 filter (CVI Laser Corporation) were placed in the laser path to remove any residual NIR emission. The light emitted by the sample was filtered with a 1100 nm long-pass filter (Edmund Optics) and later by a narrow bandpass filter at 1275 nm (BK-1270-70-B, bk Interferenzoptik). A thermoelectric-cooled NIR-sensitive photomultiplier tube assembly (H9170-45, Hamamatsu Photonics, Hamamatsu, Japan) was used as detector. Photon counting was achieved with a multichannel scaler (NanoHarp 250, PicoQuant, Berlin, Germany). The time dependence of the 1 O2 phosphorescence with the signal intensity S(t) is described by Equation (4), in which τT and τ∆ are the lifetimes of the photosensitizer triplet state and of 1 O2 respectively, and S(0) is a pre-exponential parameter proportional to Φ∆ . Curve fitting was performed with the Graphpad Prism version 7.00 for Windows, (GraphPad Software, La Jolla California USA, www.graphpad.com). Φ∆ was determined by comparing the S(0) values of optically matched solutions of the drug and the reference (phenalenone (PN), in toluene and THF Φ∆ = 0.99 and 0.96 respectively) [35,36], at 355 nm as described by Equation (5). τ∆  t − t  − S(t) = S(0)· · e τ∆ − e τT (4) τ ∆ − τT S(0)S ΦS∆ = ΦR∆ · (5) S(0)R 3.4. Determination of the Log Po/w Initially, equal volumes of n-octanol and water were mixed vigorously for 3 days at 25 ◦ C to promote solvent saturation in both phases. Each sample of Zn(II)Pc 1 was then added from stock solutions in DMSO to 2 mL of the mixture (%DMSO < 1%, [Zn(II)Pc] = 10–5 M) and stirred for 30 min; next, they were incubated 1 h at room temperature. After separation, 10 µL of n-octanol phase and 10 µL of PBS phase were taken and diluted by DMSO to 1.01 mL. The UV–Vis spectra of both phases were recorded, and the partition coefficient was calculated based on the absorbance values at Q-band maxima (702 nm for ABAB-1, 685 nm for A3 B-1 and 681 nm for A4 -1). The results are the average of three independent measurements. ! A(DMSO)O ·VW logPOW = log A(DMSO)W ·VO 3.5. Biological Assays 3.5.1. Cell Culture HeLa (cervical adenocarcinoma cells) and SCC-13 (skin squamous carcinoma cells) human cancer cell lines were used for cell cultures. They were grown using DMEM (Dulbecco’s Modified Eagle’s medium) culture media, supplemented with 10% Foetal Bovine Serum (FBS) and 1% antibiotics (G penicillin, 100 U/mL; streptomycin, 100 µg/mL), purchased from Fisher. They were incubated in HERACell incubator (Heraus) at 37 ◦ C with 5% CO2 concentration and 95% relativity humidity. 3.5.2. Photodynamic Treatment Solutions of ABAB-1, A3 B-1, and A4 -1 were prepared in concentrations of 1·10−6 and 1·10−7 M and added to cell cultures in well plates P24 with DMEM (supplemented with antibiotics but without FBS). The cells were incubated with each Pc (previously dissolved in DMSO keeping the DMSO content below 1%) for 5 h in darkness, immediately were irradiated with different red light doses (3, 6 and 9 J/cm2 ). Light source was constituted by a rectangular matrix with 384 LED (light emitting diodes), Molecules 2020, 25, 213 14 of 18 (WP7143 SURC, Kingbright) with an emission peak of 637 nm and a bandwidth of ± 17 nm. The fluence rate used was 12.7 mW/cm2 . After irradiation, cell culture was replaced by fresh media supplemented with antibiotics and FBS, and cells were incubated 24 h until their evaluation. 3.5.3. Cell Viability Cell survival was determined after photodynamic treatment, both darkness plates and irradiated plates, through MTT assays. This is a colorimetric technique based on mitochondrial enzymes capacity of alive cells to reduce the water-soluble compound 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan, an insoluble compound which has a purple colour. After 24 h of incubation, MTT was added to well plates in a final concentration of 100 µg/mL, for 3 h at 37 ◦ C. Then, DMSO (Panreac) was added in order to solve formazan and optical density was measured in SpectraFluor plate reader (Tecan), with a wavelength of 542 nm. 3.5.4. Measurement of Intracellular ROS The intracellular production of ROS in SCC-13 and HeLa cells was evaluated as previously described [37]. Cells were incubated with Zn(II)Pcs (1·10−6 M) for 5 h, and during the last hour 2,7-dichloro-dihydrofluorescein diacetate (DHF-DA, Abcam) was added to the cultures, reaching a final concentration of 6·10−6 M. Afterwards, and without removing DHF-DA, cells were exposed to green light (3 and 6 J/cm2 ) and immediately after irradiation analyzed by fluorescence microscopy, under blue excitation light (λexc = 436 nm). ROS production was quantified by using Image J after measuring green fluorescence. 3.5.5. Optical Microscopy and Statistical Analysis Microscopic observations were carried out using an Olympus BX61 epifluorescence microscope equipped with green filter (exciting filter BP510-550). Photographs were obtained with the digital camera Olympus CCD DP70 and processed using the Adobe Photoshop CS5 extended version 12.0 software (Adobe Systems Inc., San Jose, CA, USA). Data are expressed as the mean value of at least three experiments ± standard deviations (SD). The statistical significance was determined using analysis of variance (ANOVA) followed by Bonferroni’s test, and p < 0.05 was considered statistically significant. 3.5.6. Subcellular Localization In order to determine intracellular localization of Zn(II)Pcs, cells were grown in coverslips in P12 well plates and they were incubated with the PS in a concentration of 10−6 M for 18 h at 37 ◦ C. After incubation, cells were washed with PBS and were observed with fluorescence microscopy. 4. Conclusions and Future Perspectives Three amphiphilic polyethyleneglycol-functionalized Zn(II)Pcs, namely, ABAB-1, A3 B-1, and A4 -1, have been synthesized in the search for new PS for PDT applications. ABAB-1, A3 B-1 are both obtained in a crossed-condensation reaction of two differently substituted phthalonitriles, combining bulky fluorinated substituents in the B isoindoles, which have previously proved effective in suppressing aggregation and increasing the 1 O2 generation efficiency, and hydrophilic triethyleneglycol substituents in A isoindoles to impart water solubility. On the other hand, A4 -1 is an octa(triethyleneglycol) Pc which has been prepared to experimentally demonstrate that the presence of the bulky fluorinated substituents is fundamental to achieve good photosensitization abilities. In fact, photophisycal and aggregation studies have determined that ABAB-1 is the compound that most efficiently generates 1 O (τ trend is ABAB-1 > A B-1 > A -1), and is less prone to aggregate in water media. Finally, 2 ∆ 3 4 in vitro assays have been carried out in order to determine the toxicity for tumor cells for the three Zn(II)Pcs. It has been proved that none of these Pcs are toxic for tumor cells in darkness, but when Molecules 2020, 25, 213 15 of 18 the tissue is irradiated in the presence of the PS, fatality turns out. Moreover, consistently with the 1O generation studies, the impact is higher in the case of ABAB-1 and A3 B-1, than A4 -1. These 2 findings make Zn(II)Pcs with bulky bis(trifluoromethyl)phenyl groups, particularly those with an ABAB pattern candidates of choice as PS. The next step in this research is to incorporate groups with extended hydrophilicity, and also bind molecules for specific targeting that would allow increasing selectivity against different tumor tissues. Molecules 2019, 24,Materials: Supplementary x FOR PEERThe REVIEW following are available online. 15 of 17 Author Author Contributions: Chemical synthesis, Contributions: Chemical synthesis,M. M.Á.R.-M., Á. R.-M., and and A.G.C.; A. G. C.;photophysical photophysicaland and aggregation aggregation studies: studies: M.Á.R.-M.; biological studies, M.M. and P.G.-J.; writing—review and editing, M.Á.R.-M. M. Á. R.-M.; biological studies, M. M. and P. G.-J.; writing—review and editing, M. Á. R.-M. and G.d.l.T.; andsupervision, G. de la T.; project administration, supervision, project administration, and fundingÁ.J., and funding acquisition, S.N., G.d.l.T., acquisition, Á. J., and T.T. S. N., G.All authors de la T., andhave T. T.read and agreed to the published version of the manuscript. Funding and Funding: This acknowledgments: research was funded Thisbyresearch MINECO, wasSpain funded by MINECO, Spain (CTQ2017-85393-P (CTQ2017-85393-P and and CTQ2016-78454-C2-1-R), CTQ2016-78454-C2-1-R) , Instituto de Salud Carlos III and Feder Funds (FIS PI18/00708) and Instituto de Salud Carlos III and Feder Funds (FIS PI18/00708) and ERA-NET/MINECO EuroNanoMed2017-191 ERA-NET/MINECO /PCIN-2017-042. EuroNanoMed2017-191 / PCIN-2017-042. Conflicts Conflictsof Interest: The ofInterest: The authors authorsdeclare declareno noconflict conflictsofofinterest. interest. Appendix A Appendix A Figure A1. Figure 1. Subcellular localization Subcellular of organelles localization of SCC-13 of organelles and HeLa of SCC-13 cells after and HeLa cellsincubation with known after incubation with fluorescent known probes.probes. fluorescent Phase Phase contrast (PhC). contrast Green (PhC). fluorescence Green is is fluorescence from fromGolgi Golgiapparatus apparatus (Golgi), mitochondrial(Mito) mitochondrial (Mito)and and lysosomes lysosomes (Lyso). (Lyso). A (450–490 A blue blue (450–490 nm) exciting nm) exciting lamp waslampused was used for for organelles organelles probes. probes. Scale bar 10Scale µm. bar 10 μm. Materials Materials andandmethods methodsforfor subcellular subcellular localization localization of Figure of Figure A1: To A1: To analyze theanalyze the intracellular intracellular localization localization of organelles,ofSCC-13 organelles, SCC-13 and HeLa cellsand HeLa were cellsonwere grown grownand, coverslips on coverslips and, incubated with incubated with known known fluorescent fluorescent probes probes (LysoTracker for lysosomes for lysosomesGreen (LysoTracker DND-26, Green DND-26,mitochondria Invitrogen), Invitrogen), mitochondria (MitoTracker (MitoTracker Green Green FM, FM, Invitrogen), Invitrogen), or Golgi apparatus or (NBD, Golgi C6-ceramide apparatus (NBD, C6-ceramide (N-[6-[(7-nitro-2-1,3- (N-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-D-erythro-sphingosine, benzoxadiazol-4-yl)amino]hexanoyl]-D-erythro-sphingosine, Invitrogen)) at the Invitrogen)) at the concentrations concentrations indicated by theindicated byThen, suppliers. the suppliers. cells wereThen, cells briefly were briefly washed in PBS,washed in PBS, mounted mounted on slides with aondrop slidesof with a drop PBS and of PBS andobserved immediately immediately observed under under themicroscope. the fluorescence fluorescence microscope. 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