nanomaterials Article Photoactivable Ruthenium-Based Coordination Polymer Nanoparticles for Light-Induced Chemotherapy Junda Zhang 1 , Vadde Ramu 2 , Xue-Quan Zhou 2 , Carolina Frias 1 , Daniel Ruiz-Molina 1 , Sylvestre Bonnet 2, * , Claudio Roscini 1, * and Fernando Novio 1, * 1 Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and BIST, Campus UAB, Bellaterra, 08193 Barcelona, Spain; junda.zhang@icn2.cat (J.Z.); caarolina.frias@gmail.com (C.F.); dani.ruiz@icn2.cat (D.R.-M.) 2 Leiden Institute of Chemistry, Universiteit Leiden, Einsteinweg 55, 2333 CC Leiden, The Netherlands; ramuvadde1@gmail.com (V.R.); x.zhou@lic.leidenuniv.nl (X.-Q.Z.) * Correspondence: bonnet@chem.leidenuniv.nl (S.B.); claudio.roscini@icn2.cat (C.R.); fernando.novio@icn2.cat (F.N.) Abstract: Green light photoactive Ru-based coordination polymer nanoparticles (CPNs), with chemi- cal formula [[Ru(biqbpy)]1.5 (bis)](PF6 )3 (biqbpy = 6,60 -bis[N-(isoquinolyl)-1-amino]-2,20 -bipyridine; bis = bis(imidazol-1-yl)-hexane), were obtained through polymerization of the trans-[Ru(biqbpy) (dmso)Cl]Cl complex (Complex 1) and bis bridging ligands. The as-synthesized CPNs (50 ± 12 nm di- ameter) showed high colloidal and chemical stability in physiological solutions. The axial bis(imidazole) ligands coordinated to the ruthenium center were photosubstituted by water upon light irradiation in   aqueous medium to generate the aqueous substituted and active ruthenium complexes. The UV-Vis spectral variations observed for the suspension upon irradiation corroborated the photoactivation Citation: Zhang, J.; Ramu, V.; of the CPNs, while High Performance Liquid Chromatography (HPLC) of irradiated particles in Zhou, X.-Q.; Frias, C.; Ruiz- physiological media allowed for the first time precisely quantifying the amount of photoreleased com- Molina, D.; Bonnet, S.; Roscini, C.; Novio, F. Photoactivable plex from the polymeric material. In vitro studies with A431 and A549 cancer cell lines revealed an Ruthenium-Based Coordination 11-fold increased uptake for the nanoparticles compared to the monomeric complex [Ru(biqbpy)(N- Polymer Nanoparticles for methylimidazole)2 ](PF6 )2 (Complex 2). After irradiation (520 nm, 39.3 J/cm2 ), the CPNs yielded up to a Light-Induced Chemotherapy. two-fold increase in cytotoxicity compared to the same CPNs kept in the dark, indicating a selective Nanomaterials 2021, 11, 3089. https:// effect by light irradiation. Meanwhile, the absence of 1 O2 production from both nanostructured and doi.org/10.3390/nano11113089 monomeric prodrugs concluded that light-induced cell death is not caused by a photodynamic effect but rather by photoactivated chemotherapy. Academic Editor: Tambet Teesalu Keywords: coordination polymer nanoparticles; nanoparticles; photoactivated chemotherapy; prodrug; Received: 13 October 2021 drug delivery; ruthenium-based drug Accepted: 12 November 2021 Published: 16 November 2021 Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in published maps and institutional affil- Ruthenium-based drugs have raised interest over the last years as an alternative iations. to Pt drugs for oncotherapy, with an increasing number of them entering clinical trials, such as NAMI-A, KP 1019, KP 1339, or TLD–1433 [1–7]. Especially relevant has been the development of ruthenium prodrug molecular complexes bearing photolabile ligands for photoactivated chemotherapy (PACT) applications [8–12]. Interestingly, these complexes generally exhibit low toxicity in the dark but become toxic once activated by visible light Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. irradiation. The mechanism of prodrug activation is related to the specific ligand photo- This article is an open access article substitution by water molecules, to afford activated aqua photoproducts able to induce distributed under the terms and a therapeutic action [10,13–17]. Moreover, PACT is an oxygen-independent activation conditions of the Creative Commons mechanism that works even under hypoxic conditions. This feature makes it potentially Attribution (CC BY) license (https:// more versatile than type II photodynamic therapy (PDT), which requires the presence of creativecommons.org/licenses/by/ a significant amount of dioxygen to generate enough reactive singlet oxygen species to 4.0/). induce cytotoxicity [10,11,14,18–24]. Nanomaterials 2021, 11, 3089. https://doi.org/10.3390/nano11113089 https://www.mdpi.com/journal/nanomaterials Nanomaterials 2021, 11, x FOR PEER REVIEW 2 of 17 Nanomaterials 2021, 11, 3089 2 of 16 a significant amount of dioxygen to generate enough reactive singlet oxygen species to induce cytotoxicity [10,11,14,18–24]. However, before However, before reaching reaching clinical clinical use, use, photoactivated photoactivatedRu Rucomplexes complexesmust mustfacefacekeykey challenges such challenges suchasaswaterwatersolubility, solubility, preferential preferential accumulation accumulation in tumors [25], precise in tumors con- [25], precise trolled release controlled of the release drug of the [26–28], drug [26–28],increase in the increase in biocompatibility the biocompatibility while minimizing while minimizing re- sidual toxicity residual toxicityin the in thedarkdark[29–31], and improvement [29–31], and improvement of theiroffast clearance their from thefrom fast clearance blood- the stream [32]. To bloodstream [32].overcome To overcome most most of these limitations, of these photolabile limitations, complexes photolabile complexes[33,34],[33,34], and specifically and Ru-based specifically Ru-based complexes [35], can complexes [35],becanincorporated be incorporated in nanoparticles in nanoparticles(NPs) for(NPs)their for application their in photoinduced application in photoinduced therapies. For instance, therapies. Wu et al.Wu For instance, have et reported al. have the covalent reported the link of Ru covalent linktoof block Ru tocopolymers block copolymers[10,36] that [10,36]stabilize photoactivatable that stabilize ruthenium photoactivatable com- ruthenium plexes under complexes physiological under physiological conditions conditions [9].[9]. This This strategy strategy includes includesruthenium-containing ruthenium-containing block copolymer block copolymerunits unitsthatthatself-assembled self-assembled into nanoparticles into nanoparticles in aqueous in aqueous solution with with solution ex- cellent uptake excellent uptake in vitro in vitroandandin vivo results. in vivo The inhibition results. The inhibitionof cancer cells was of cancer related cells to the was related generation to of singlet the generation oxygen of singlet (1O2) upon oxygen irradiation (1 O2 ) upon with red irradiation withlight [37,38]. red light OtherOther [37,38]. ap- proaches involve approaches involve thetheconjugation conjugation of photocleavable of photocleavable Ru complexes Ru complexes to thetosurface of up-of the surface converting NPs upconverting NPs [39–42]. [39–42]. Even so, so, Even thethe encapsulation encapsulation of photoactive of photoactive Ru-based Ru-based complexes complexes is inin is itsits fledgling fledgling stage, stage, sosothere thereisisa agrowing growinginterest interesttotodevelop developnovel novelNPs NPsthat thatallow allowa a proper fine-tune proper fine-tunestructure/function structure/functioncorrelations correlationsand andadaptadaptit it forfor theirtheir useuse in photoacti- in photoactivated vated chemotherapy chemotherapy [43]. [43]. Herein, we Herein, wehypothesize hypothesizethat thatcoordination coordination polymer polymer nanoparticles nanoparticles (CPNs) bearing (CPNs) Ru- bearing active complexes Ru-active complexes as constitutive as constitutive building building blocks blocks and anda aphotocleavable photocleavablebridging bridgingligand ligand may represent may represent aastep stepforward forwardforfor PACTPACT applications applications (the(the schematic schematic representation representation of theof proposed the proposed system systemis shown is shownin Figure in Figure1). In1).addition In additionto achieve smart smart to achieve NPs with NPshigh withpay- high loads, CPNs payloads, CPNshavehavealready beenbeen already successfully successfullydemonstrated demonstratedto be highly performing to be highly as bi- performing ocompatible as biocompatible contrast agentsagents contrast and antitumoral and antitumoral application, including application, Pt (IV)-based including CPNs Pt (IV)-based [44,45].[44,45]. CPNs These nanoformulations These nanoformulations offer good colloidal offer stability, scalability, good colloidal cellular internal- stability, scalability, cellular ization, and evenand internalization, more evennoticeably high payloads, more noticeably as the prodrug high payloads, as theconstitutes the backbone prodrug constitutes the of the nanoparticles backbone polymer itself of the nanoparticles polymer [46].itself All these [46]. advantages turn out to All these advantages be really turn out to helpful be really to reduce helpful tothe dose,the reduce the dose, irradiation intensity required the irradiation intensitytorequired activate the anticancer to activate thedrug diffu- anticancer sion, and drug therefore diffusion, andany side effect. therefore any Though, side effect. as far as we know, Though, as far as thewe number know,of theruthenium- number of based coordinationcoordination ruthenium-based polymers with antitumor polymers withapplications antitumorisapplications rather limited, none oflimited, is rather them being photoactivable as far as we know. none of them being photoactivable as far as we know. Figure 1. Scheme of synthesis and photoactivation process of Ru-based coordination polymer nanoparticles (RuBIS Figure 1. Scheme of synthesis and photoactivation process of Ru-based coordination polymer nanoparticles (RuBIS CPNs). CPNs). We have achieved this challenge with the synthesis of CPNs containing [Ru(biqbpy) We havemonomer (dmso)Cl]Cl achieved(complex this challenge withbiqbpy 1, where the synthesis = standsoffor CPNs containing [Ru(biq- 6,60 -bis[N-(isoquinolyl)-1- bpy)(dmso)Cl]Cl 0 monomer (complex 1, where biqbpy = stands for 6,6′-bis[N-(isoquinolyl)- amino]-2,2 -bipyridine), which is known to form cytotoxic aqueous active species [Ru(biqbpy) 1-amino]-2,2′-bipyridine), (H 2+ which is known to form cytotoxic aqueous active species 2 O)2 ] upon blue or green light activation [14]. The polymerization process was performed [Ru(biqbpy)(H 2O)2]2+ upon blue or green light activation [14]. The polymerization process using the photocleavable bis(imidazol-1-yl)-hexane (BIS) ligand and following a methodology previously described for the synthesis of non-photoactive CPNs of relevance in biological applications [47–50]. Nanomaterials 2021, 11, 3089 3 of 16 2. Materials and Methods 2.1. Reagents and Instrumentation Solvents were purchased from Sigma–Aldrich (Merck KGaA, Darmstadt, Alemania) and used as received, and complex 1 was synthesized and characterized according to previously reported methodology [14]. Fourier transform infrared (FTIR) spectra were carried out with a Tensor 27/PMA50FTIR Spectrometer (Bruker Optics GmbH, Ettlingen, Germany) in a range of 4000–400 cm−1 . Determination of the particle-size distributions and the zeta potential values were measured by dynamic light scattering (DLS) using a ZetaSizer nano ZS (ZEN3600, Malvern Instruments, Ltd., Malvern, UK). Scanning Electron Microscopy (SEM) images were obtained on a scanning electron microscope (FEI Quanta 650 FEG, Thermo Fisher Scientific, Eindhoven, The Netherlands). The samples were casted on aluminum holders following by evaporation, and later, a thin platinum layer was sprayed to increase the conductivity of samples. Ultraviolet–visible spectroscopy (UV-vis) study was carried out in the Agilent Cary 60 spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) in the dark using a 1 cm quartz cuvette with a stirring bar, containing 2 mL of RuBIS CPNs (20 µg/mL). Time-dependent UV-Vis spectra during irradiation were recorded at regular time intervals (specified in the spectra) after irradiation of the stirred sample with a continuous beam of a green (λ = 532 nm, 30 mW, 0.42 mW/cm2 ) or blue laser (450 nm, 100 mW, 0.35 mW/cm2 ) set in front of the cuvette. 2.2. Synthesis and Characterization of the Photoactive Materials 2.2.1. Synthesis of Complex 2 [Ru(biqbpy)(dmso)Cl]Cl (50.0 mg, 0.072 mmol) was dissolved in EtOH and H2 O (5 mL, v/v = 1/1). Then, 1-methylimidazole (60.0 mg, 0.73 mmol) was injected in the mixture and refluxed for 12 h under N2 protection. The reaction mixture was cooled to room tempera- Nanomaterials 2021, 11, x FOR PEER REVIEW 4 of 17 ture. Then, ice-cold water (5 mL) and KPF6 (20 mg) were added to the reaction mixture; thus, dark brown precipitate was formed and filtered. After column chromatography (SiO2 , Ethyl acetate/MeOH = 10:1), 2 (50 mg, 80%) [Ru(biqbpy)(N-methylimidazole)2 ] (PF6 )2 results and complex discussion (complex section, 2, Figure enabled 2a) was us toaspropose obtained the chemical a dark brown formula solid. For [[Ru(biq- NMR and mass bpy)]1.5(bis)](PF spectrometry 6)3 (Figure 2b). see Figures S1–S4. characterization, Figure 2. Figure 2. Chemical Chemical representation representation of of complex complex 22 (a) (a) and and RuBIS RuBIS CPNs CPNs (b). (b). 1 H NMR (360 MHz, (CD ) SO) δ 10.73 (s, 2H), 8.93 (d, J = 8.1 Hz, 2H), 8.46 (d, 2.3. HPLC Methodology for RuBIS 3 2CPNs Releasing Quantification J = 7.7 Hz, 2H), 8.21 (d, J = 6.7 Hz, 2H), 8.12 (t, J = 8.0 Hz, 2H), 7.93 (ddd, J = 23.1, 18.0, 7.3 Hz,Time-dependent 8H), 7.41 (d, J HPLC = 6.6 Hz,evolution under 2H), 7.32 irradiation (s, 2H), 6.86 (s,of2H), a RuBIS colloidal 6.08 (s, suspension 2H), 3.35 (s, 6H). (200 13 µg/mL) was performed with stirring, and aliquots at different C NMR (91 MHz, (CD3 )2 SO) δ 156.28, 151.20, 150.01, 145.50, 140.25, 135.81, irradiation times were 135.58, taken, filtered, and analyzed by HPLC. A calibration curve was performed 131.80, 129.28, 128.82, 127.86, 123.77, 122.69, 120.21, 118.81, 116.06, 115.79, 34.22. MS-using different concentrations ESI (m/z): [M]+(0.1, 0.5, 1, Calcd. for5,C10, and 20 μg/mL) + of a stock solution in PBS buffer of the 36 H32 N10 Ru 705.8, found 705.2. Elem. Anal. Calcd. for [Ru(biqbpy)(H 2O)2] activated complex. The stock solution was obtained from the irradi- 2+ C36 H32 F12 N10 P2 Ru·3H2 O: C, 40.55; H, 3.60; N, 13.51 Found: C, 40.72; H, 3.62; N, 12.95. ation with green light (100 mW, 1.1 mW/cm2) of complex 1 (1 mg/mL) dissolved in PBS solution 2.2.2. for 20 of Synthesis h to make sure that Coordination Ru complex Polymer was fully Nanoparticles RuBIS converted to the active form through Ru complex ([Ru(biqbpy)(dmso)Cl]Cl) (10.9 mg, 15.8 µmol)measurements the photocleavage and photosubstitution process. The were car- was firstly dissolved in aried out using 2-necked an HPLC Waters round-bottom flask (102695 mL)separation in 1 mL ofmodule Milli-Q (Waters ® Corp., reflux water under Milford, andMA, N2 USA) coupled to a Waters 2487 UV-Vis detector (Waters Corp., Milford, MA, USA) and using a Restek® C-18 (250 mm × 4.6 mm) column (Restek Corp., Bellefonte, PA, USA). Eluent A was a 0.1% (v/v) H3PO4 aqueous solution, and eluent B was acetonitrile absolute (HPLC grade). Before the analysis, the column was pre-equilibrated using the starting Nanomaterials 2021, 11, 3089 4 of 16 atmosphere for 10 min. BIS (3.4 mg, 15.8 µmol) was dissolved in 1 mL of Milli-Q® water and injected to the reaction slowly, and the color of the reaction changed from orange to dark brown. The reaction was stirred at 80 ◦ C at 600 rpm for 1 h in dark conditions. After 1 h, 0.5 mL of saturated KPF6 water solution was added to the mixture, causing the precipitation of a solid. The solid was purified through three times centrifugation (10 min, 4300 rpm) and washed with Milli-Q® water. Finally, the as-obtained solid was freeze-dried and stored as a powder (13.0 mg, yield = 92.8 wt %). Chemical analysis, detailed in the results and discussion section, enabled us to propose the chemical formula [[Ru(biqbpy)]1.5 (bis)](PF6 )3 (Figure 2b). 2.3. HPLC Methodology for RuBIS CPNs Releasing Quantification Time-dependent HPLC evolution under irradiation of a RuBIS colloidal suspension (200 µg/mL) was performed with stirring, and aliquots at different irradiation times were taken, filtered, and analyzed by HPLC. A calibration curve was performed using different concentrations (0.1, 0.5, 1, 5, 10, and 20 µg/mL) of a stock solution in PBS buffer of the [Ru(biqbpy)(H2 O)2 ]2+ activated complex. The stock solution was obtained from the irradiation with green light (100 mW, 1.1 mW/cm2 ) of complex 1 (1 mg/mL) dissolved in PBS solution for 20 h to make sure that Ru complex was fully converted to the active form through the photocleavage and photosubstitution process. The measurements were carried out using an HPLC Waters 2695 separation module (Waters Corp., Milford, MA, USA) coupled to a Waters 2487 UV-Vis detector (Waters Corp., Milford, MA, USA) and using a Restek® C-18 (250 mm × 4.6 mm) column (Restek Corp., Bellefonte, PA, USA). Eluent A was a 0.1% (v/v) H3 PO4 aqueous solution, and eluent B was acetonitrile absolute (HPLC grade). Before the analysis, the column was pre-equilibrated using the starting conditions (99% A (v/v)) for 10 min, followed by a gradual decrease in A from 100% to 40% (v/v) in the first 20 min and lasting 5 min. Then, the mobile phase reduced to 20% A (v/v) in 1 min and lasted 4 min. At the end, mobile phase was increased to 100% A (v/v) in 1 min to elute residues, and this ratio was kept for additional 5 min. For the next injection, the mobile phase was reset to the initial conditions (A:B) 100:0 (v/v) and kept for 10 min to equilibrate. The flow rate was set at 1.0 mL/min at temperature. This method was used for both the calibration curve and quantification of an active complex release from RuBIS CPNs. 2.4. Quantitative 1 H NMR and 19 F NMR for Component Analysis of RuBIS CPNs The slow rotational correlation time in NMR of these nanoparticles in a colloidal solution makes it difficult to obtain a quantitative NMR spectrum. To accomplish this, the RuBIS CPNs were dissolved in deuterated dimethyl sulfoxide solvent ((CD3 )2 SO) containing a minimum quantity of deuterium chloride (DCl) to decompose the nanoparti- cles into the molecular entities and thus obtain sharp signals that allow quantifying the ligand-to-ligand ratio. To make sure that different spectra are comparable, the same ratio DCl/(CD3 )2 SO was used (50 µL DCl/mL (CD3 )2 SO). In addition, the internal reference (CH2 FCN) that has a hydrogen and fluorine atom was chosen to increase the accuracy. The formula shown below was used to calculate the amount of components. Ssample Nstd mstd Msample Psample = × × × × Pstd Sstd Nsample msample Mstd where S = Integrated area of the peak, N = Number of protons atoms in the functional group), m = Weighted mass, M = Molecular weight, and P = Purity. 2.5. In Vitro Studies 2.5.1. Cell Culturing Human epidermoid carcinoma A431 and human lung carcinoma A549 cancer cell lines were tested. These cell lines were distributed by the European Collection of Cell Cultures (ECACC, Salisbury, UK) and purchased through Sigma Aldrich (Merck KGaA, Nanomaterials 2021, 11, 3089 5 of 16 Darmstadt, Alemania). Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) “complete” (i.e., DMEM with phenol red, supplemented with Fetal Calf Serum (FCS, 10.0% v/v), Penicillin–Streptomycin (PS solution; 0.2% v/v), and GlutaMax (GM, 0.9% v/v)). Both cell lines were cultured under humidified conditions (37 ◦ C atmosphere containing 7.0% CO2 ) in 75 cm2 flasks and sub-cultured (1:3–1:6) upon reaching 70–80% confluency (once per week). Media were refreshed every 3 days; cells were passaged for 4–8 weeks maximum. 2.5.2. Cell-Irradiation Setup The same cell-irradiation system was used as published previously from our group [51] that consisted of a Ditabis thermostat (980923001) fitted with two flat-bottomed microplate thermoblocks (800010600) and a 96-LED array fitted to a standard 96-well plate. The λexc = 520 nm LED with power density (10.9 mW/cm2 ) (OVL-3324), fans (40 mm, 24 V DC, 9714839), and power supply (EA-PS 2042-06B) were obtained from Farnell. 2.5.3. Cytotoxicity Assay Cells were seeded at a density of 5 × 103 /mL for A549 and 8 × 103 /mL for A431 in 96-well plates at t = 0 h using Opti-MEM complete without phenol red (100 µL) and incubated for 24 h at 37 ◦ C, under 7% of CO2 . Subsequently, aliquots (100 µL) of six different concentrations between 50 ng/mL and 50 µg/mL (0.7 ng/mL to 2.83 µg/mL based on metal content) of freshly prepared stock RuBIS CPNs suspension or complex 2 solution in Opti-MEM were added to three adjacent wells as a triplicate. A minimum amount of DMSO (<0.5%) was used to dissolve the compounds, which does not harm the cells in each well, including in the control wells. After incubation in the dark for an additional 24 h, the plates were irradiated for 60 min with green light (λexc = 520 nm, power density 10.92 mW/cm2 , light dose = 39.3 J/cm2 ). After irradiation, the plates were incubated in the dark for an additional 48 h either in normoxia or hypoxic incubator. Then, the cells were fixed using cold (4 ◦ C) TCA (10% w/v; 100 µL). Subsequently, TCA was removed from the wells, and the plates were washed with water (×5), stained with SRB (0.6% w/v in acetic acid (1% v/v; 100 µL) for 30 min, washed with acetic acid (1% v/v; ≈300 µL), and air dried overnight. After solubilizing the SRB dye with Tris-base (10 mm; 200 µL), the absorbance was read in each well at λ = 510 nm by using a M1000 Tecan Reader. The fraction of viable cells in each well was calculated using SRB absorbance (Excel and GraphPad Prism software) obtained from triplicates for each concentration. The relative cell viabilities were obtained by dividing the average absorbance of the treated wells by that observed in the untreated wells for three independent biological replicates (three different passage numbers per cell line). The average cell viability was plotted versus log (concentration) (µM), including the standard deviation error of each point. The effective concentration (EC50 ) was calculated by using the dose–response curve for each cell line (dark vs. irradiated conditions), by fitting the curves to a non-linear regression function, as relative cell viability, and obtaining a variable Hill slope from Equation (1) Y = 100/(1+10((log10 EC50 −X) · Hill Slope)). (1) 2.5.4. Cellular Uptake Measurements A431 cells (5 × 105 ) were seeded in 12-well plates, incubated for 24 h under normoxic conditions, and treated with RuBIS CPNs (25 µg/mL) or complex 2 (19 µg/mL) for 2 h. Then, cells were washed thrice with cold (4 ◦ C) PBS (3 × 2 mL) to remove any compound attached outside the cells. Then, the cells were trypsinized and collected into a 2 mL Eppendorf tube in Opti-MEM media. Cells were counted on a BioRad cell-counting board and carefully washed once with cold PBS to remove trypsin. Then, collected cells were centrifuged at 3000 rpm for 5 min. The resulting cell pellet was digested using 65% HNO3 at 100 ◦ C overnight in a hot air oven. Once back to room temperature, the total volume was completed to 10 mL using Milli-Q®water. The ruthenium content and the standard deviation values of these solutions were analyzed on the duplicate experimental results Nanomaterials 2021, 11, 3089 6 of 16 using the Perkin Elmer NexION 2000 (PerkinElmer, Shelton, CT, USA) of an inductively coupled plasma mass spectrometer (ICP-MS, PerkinElmer, Shelton, CT, USA). 2.5.5. Endocytosis Inhibition Studies A431 cells (5 × 105 cells) were seeded in 12-well plates, incubated for 24 h under normoxic conditions, and then treated with NaN3 (active uptake inhibitor, 15.4 mM), NH4 Cl (20 mM), or Dynasore (dynamin-dependent endocytosis inhibitor, 80 µM) for 1 h; alternatively, the cells were incubated at 4 ◦ C for 1 h. After that, the cells were incubated with either RuBIS CPNs (50 µg/mL) or complex 2 (38 µg/mL) for 3 h in the regular incubator for the inhibitor samples or at 4 ◦ C for the low temperature samples. Then, the cells were treated as in the normal cellular uptake study. 2.5.6. ICP-MS Analysis The sample was digested in nitric acid (65%, Suprapur® , Merck, Darmstadt, Germany), while diluted (1%) nitric acid was used as a carrying solution. NIST-traceable 1000 mg/L elemental standards (TraceCERT® , Fluka Chemie GmbH, Buchs, Switzerland) were used for the calibration and as internal standards. Calibration standards were prepared in a Secuflow fume hood (SCALA, Wangen, Germany) to prevent contamination, and MiliQ® was used in all sample preparation and analysis steps. The measurements were analyzed using the NexION® 2000 ICP-MS (PerkinElmer, Shelton, CT, USA) equipped with a concentric glass nebulizer and Peltier-cooled glass spray chamber. An SC2 DX autosampler (PerkinElmer, Shelton, CT, USA) was used for sample introduction. Data recording and processing was done by using SyngistixTM Software (v.2.5, PerkinElmer, Shelton, CT, USA). Trace elemental calibration standards were prepared at 0, 1, 4, 20, and 100 µg/L using an NIST-traceable 1000 mg/L Ru standard. An additional set of calibration for Ru (0, 0.1, 0.5, 2.5, and 10 µg/L) was prepared for samples that were anticipated to contain low-level Ru. Samples were analyzed without dilution to minimize the possibility of contamination, using 10 µg/L Rh and In as the internal standard. To check the calibration, samples were analyzed through a repeated measurement of one of the calibration standards and a blank measurement. Curves with correlation coefficient higher than 0.999 were accepted for the calibration. 2.5.7. Singlet Oxygen (1 O2 ) Production Studies Singlet oxygen generation measurements were conducted in cell-growing medium using 9, 10-anthracenediyl-bis(methylene) dimalonic acid (ABMDMA) as an 1 O2 -specific probe. ABMDMA is a hydrophilic anthracene derivative that reacts with 1 O2 to produce the corresponding endoperoxide [52], thereby lowering the absorbance at 400 nm. For the experiment, 0.1 mM of ABMDMA (in Opti-MEM) was mixed with RuBIS CPNs (25 µg/mL), which was previously dispersed in Opti-MEM cell culture media. The resulting samples were taken into a 3 mL quartz cuvette to record the absorbance in the dark or following green light irradiation (λexc = 520 nm, 39.3 J/cm2 ). Absorption spectra were recorded initially every 30 s during the first 1 min of continuous light irradiation and successively every 1 min interval during 6 min. The reference rose Bengal dye caused significant changes to the absorption spectra of ABMDMA at 400 nm, which indicated the production of 1 O2 with a quantum yield of Φ∆ = 0.68 [53]. 3. Results and Discussion 3.1. Synthesis and Characterization In a typical synthesis, complex 1 was refluxed in 1 mL of MilliQ® water under N2 atmosphere. Subsequently, 1 mL of ethanol solution containing one equivalent of BIS lig- and was slowly injected in the Ru complex solution, and the reflux was maintained for 1 h. Afterwards, the solution was cooled down to room temperature, and a saturated KPF6 solu- tion was added to the mixture, resulting in a brown precipitate. The solid was centrifuged, washed with cold ethanol, and freeze-dried for subsequent storage and characterization. Fourier-transform infrared spectroscopy (FTIR) of the freeze-dried solid showed the pres- In a typical synthesis, complex 1 was refluxed in 1 mL of MilliQ® water under N2 atmosphere. Subsequently, 1 mL of ethanol solution containing one equivalent of BIS lig- and was slowly injected in the Ru complex solution, and the reflux was maintained for 1 h. Afterwards, the solution was cooled down to room temperature, and a saturated KPF6 Nanomaterials 2021, 11, 3089 solution was added to the mixture, resulting in a brown precipitate. The solid was centri- 7 of 16 fuged, washed with cold ethanol, and freeze-dried for subsequent storage and character- ization. Fourier-transform infrared spectroscopy (FTIR) of the freeze-dried solid showed the presence ence of the peaks of the typical typicalofpeaks both of BISboth (3000BIScm (3000 cm−1,cm −1 , 1509 1509−1 cm −1 and 1472−cm and 1472 −1) [54] and cm 1 ) [54] and of of complex complex 1 (1532 1 (1532 cmcm and1098 −1−1and 1098 cmcm−−11).).More Moreimportantly, importantly,new newbandsbands at at 839839cmcm−1 and −1 and429 cm −1 assigned − 1 to antisymmetric stretching of PF 6 and Ru-N stretching modes, respectively, 429 cm assigned to antisymmetric stretching of PF6 and Ru-N stretching modes, respec- confirmed tively, the coordination confirmed the coordination of theofBISthe ligand BIS ligand to complex to complex 1 and the the 1 and presence presenceof PFof 6PFas6 −a − counterion as a counterion (Figure S5) [55]. (Figure Dynamic S5) [55]. Dynamic lightlight scattering scattering(DLS)(DLS) analysis analysis of colloidal of colloidal CPNs CPNs in Milli-Q ® water ® showed an average hydrodynamic diameter in Milli-Q water showed an average hydrodynamic diameter of 93 ± 46 nm (Figure 3a) of 93 ± 46 nm (Figure 3a) in agreement with the value of 50 ± 12 nm of average size in agreement with the value of 50 ± 12 nm of average size found using scanning electron found using scanning electron microscopy (SEM) microscopy (SEM) (Figure (Figure 3b). 3b). Moreover, Moreover, the the colloidal colloidal stability stability of of the the nanoparticles nanoparticles for for atat least 24 least 24 hhin inthe thedark darkwas wasalso alsocorroborated corroborated byby DLSDLS analysis analysis in ain20a 20 mg/mL mg/mL BSA-contain- BSA-containing ing PBS PBS solution, solution, whichwhichwaswas used used as aas a physiological physiological media media model model (Figure (Figure S6).S6). TheThe absence absence of of the the diffraction diffraction pattern pattern observed observed from from theX-ray the X-raypowder powderdiffraction diffraction(XRD) (XRD)indicated indicated the the amorphous nature amorphous nature of of as-obtained as-obtained RuBIS RuBIS CPNs CPNs (Figure (Figure S7). S7). Finally, Finally, inductively inductively coupled coupled plasma mass spectrometry (ICP-MS) analysis of the nanoparticles plasma mass spectrometry (ICP-MS) analysis of the nanoparticles showed 6.9 ± 0.2 wt showed 6.9 ± 0.2 wt % % of Ru of Ru content content (Figure (Figure S8) S8) and and H11 H and and FF nuclear 19 19 nuclear magnetic resonance (NMR) magnetic resonance (NMR) of of dissolved dissolved CPNs in CPNs in acidic acidic solvent, solvent, using using CH CH22FCN FCN as the internal as the internal reference reference (see (see the the procedure procedure in in the the Materials and Materials and methods methods section section andand NMRNMR spectra spectra in Figure S9a,b), in Figure S9a,b), which which allowed allowed us us to to propose the propose the chemical chemicalformulaformula[[Ru(biqbpy)] [[Ru(biqbpy)]1.51.5(bis)](PF (bis)](PF66))33.. The The stoichiometric stoichiometric deviation deviation from the from thetheoretical theoreticalexpected expectedratio ratioof of thethe components components for afor a linear linear polymerpolymer ([Ru(biq- ([Ru(biqbpy)]: bpy)]:BIS = 1:1) is quite archetypal for CPNs. This is attributed to the out-of-equilibrium BIS = 1:1) is quite archetypal for CPNs. This is attributed to the out-of-equilibrium synthetic synthetic conditions conditions that lead to that thelead fastto the fast precipitation precipitation process ofprocess oligomeric of oligomeric species with species with different different stoichiometry stoichiometry [45,46,48,49,56,57]. [45,46,48,49,56,57]. In any case, In the anyRu case, the Rupayload complex complex payload value of 41value wt % of is 41 wt than more % is four-fold more thanhigher four-fold thanhigher than most conventional most conventional metallodrug-loadedmetallodrug-loaded polymer carriers poly- mer carriers known to date known to date (typically less(typically than 10%) less than [58]. It is10%) worth[58]. It is worththat mentioning mentioning that the the characteriza- characterization analysis was successfully performed for at least three different batchesthe tion analysis was successfully performed for at least three different batches to assure to assure the reproducibility reproducibility of the syntheticof themethodology. synthetic methodology. Figure 3. (a) Size distribution of RuBIS CPNs, measured using dynamic light scattering in PBS solution (average diameters of 50 ± 12 nm). (b) Representative SEM image of RuBIS CPNs, scale bar: 500 nm. 3.2. Photoreactivity of RuBIS CPNs 3.2.1. Monitorization by UV-Vis UV-Vis spectroscopy of fresh-made PBS suspensions of the nanoparticles showed an absorption band with a maximum at λmax = 315 nm and two shoulders at λabs = 365 nm and 439 nm lengthening up to 600 nm (Figure 4, black curve, time = 0 min). No spectral changes were observed in the dark upon time (3 h and 10 min, Figure S10) while, as expected, significant changes were found under irradiation. For these experiments, two different irradiation wavelengths were selected: (i) 450 nm, close to the CPNs absorption maximum in the visible region (Figure S11) and (ii) 532 nm, taking advantage of the tail of the broad absorption band (Figure 4). This last wavelength should be not only suitable for triggering UV-Vis spectroscopy of fresh-made PBS suspensions of the nanoparticles showed an absorption band with a maximum at λmax = 315 nm and two shoulders at λabs = 365 nm and 439 nm lengthening up to 600 nm (Figure 4, black curve, time = 0 min). No spectral changes were observed in the dark upon time (3 h and 10 min, Figure S10) while, as expected, Nanomaterials 2021, 11, 3089 significant changes were found under irradiation. For these experiments, two different 8 of 16 irradiation wavelengths were selected: (i) 450 nm, close to the CPNs absorption maximum in the visible region (Figure S11) and (ii) 532 nm, taking advantage of the tail of the broad absorption band (Figure 4). This last wavelength should be not only suitable for triggering thephotosubstitution the photosubstitutionreactions reactionsbut butititalso alsoless lessharmful harmfulthan thanblue bluelight lighttotoliving livingcells cellswith with also deeper penetration in biological tissues. also deeper penetration in biological tissues. Figure4.4.(a) Figure (a)Evolution Evolutionofofthe theUV-Vis UV-Visspectra spectraofofa aPBS PBSsuspension suspensionofofRuBIS RuBISCPNs CPNsupon upongreen greenlight light (λ exc = 532 nm); (b) plot of absorption changes at 326 nm (black) and 439 nm (λexc = 532 nm); (b) plot of absorption changes at 326 nm (black) and 439 nm (red). (red). Irradiationwith Irradiation with450450 nmnm (0.35 (0.35 W/cmW/cm 2) for 2 ) for the the firstfirst 10 showed 10 min min showeda red ashift redofshift of the the main mainfrom band, band, from λmax λmaxto= 326 = 315 315nmto 326 and nm and an intensity an intensity increase inincrease in the at the shoulder shoulder at λnm. λabs = 439 abs = 439 nm. Irradiation for longer periods (up to 2.5 h) evidenced an intensity decrease in both Irradiation for longer periods (up to 2.5 h) evidenced an intensity decrease in both bands, in- bands, the cluding including theatshoulder shoulder λabs = 365 atnm λabsuntil = 365itsnm until its disappearance disappearance (Figure S11)(Figure and anS11) and an isosbestic isosbestic point at λisopoint = 406atnm. λisoThis = 406 nm. This time-dependent time-dependent two-step two-step process wasprocess was explained tentatively tentatively by an initial fast formation of photoinduced intermediate species (tirradiation ≤ 10 min) and a subsequent slower photochemical reaction (tirradiation > 10 min) leading to the forma- tion of the final photoproduct. Irradiation with green light (λexc = 532 nm, 0.42 W/cm2 ) yielded similar results, although the spectral changes occurred more slowly due to the lower absorption of the RuBIS CPNs at the used wavelength. A red shift from λmax = 315 to 326 nm and an increase in intensity at λabs = 439 nm was observed for the first 15 min of irradiation, while longer irradiation periods (up to 3 h) induced a decrease in the two bands (326 nm and 439 nm) and the formation of an isosbestic point (Figure 4). Interestingly, similar evolution in UV-Vis spectra was observed for the molecular complex 1 under both blue and green light irradiation (Figure S12) [14], which suggests a similar photo-induced process toward the active photoproduct. Nanomaterials 2021, 11, 3089 9 of 16 3.2.2. HPLC Studies Complementary high-performance liquid chromatography (HPLC) studies were done to study the photoproducts resulting upon RuBIS CPNs green light irradiation. Its intrinsic selectivity and sensitivity compared to 1 H-NMR or UV-Vis spectroscopy allows the precise quantification of photoproducts as well as the ability to differentiate final Ru-containing by- products more specifically than inductively coupled plasma mass spectrometry (ICP-MS). Before any measurement with RuBIS CPNs, a stock solution in PBS buffer of complex 1 (1 mg/mL), was irradiated with green light (1.1 W/cm2 ) for 20 h (Figure S13). Elution of the irradiated sample resulted in a single peak at 18.3 min assigned to the active com- plex [Ru(biqbpy)(H2 O)2 ]2+ , as confirmed by ESI mass spectrometry (Figure S14) with the corresponding m/z = 271.0 (calc. m/z = 270.8). As complex 1 to active form conversion was quantitative under such experimental conditions [14], a calibration curve of the fully activated complex was obtained at different concentrations (R2 = 0.996) (Figure S15). Subsequently, a colloidal suspension of RuBIS CPNs in PBS buffer was irradiated (532 nm, 1.1 W/cm2 and 24 h), and aliquots at different irradiation times were taken, filtered, and analyzed by HPLC. To guarantee the detection of the complex released, the concentration of the initial suspension was increased with respect to that used in UV-Vis experiments up to 200 µg/mL and the irradiation time was enlarged. The results are shown in Figure 5. After the first 30 min, three different peaks with retention times of 14.7, 16.9, and 18.3 min, associated to different species, appeared after 30 min (Figures 5a and S16). Further irradiation up to 6 h induced a notable increase in the peak at 18.3 min, while the two other decreased until almost complete disappearance after 20 h (Figure 5b). The formation of intermediate species at shorter irradiation times and their decrease upon prolonged irradiation to form a final product resembles the behavior observed with UV- Vis experiments (the different conversion times observed by UV-Vis vs. HPLC for the intermediates and final photoproduct were ascribed to the different CPNs concentrations used in each case). To get more detailed information on the intermediate as well as the final photoproduct chemical composition, mass spectrometry was used. Analysis of intermediate fractions at 14.7 and 16.9 min revealed oligomeric species that may come from larger fragments, such as {[Ru2 (biqbpy)2 bis(MeOH)2 ](PF6 )2 }2+ (found m/z = 828.8 calc. m/z = 828.7) (Figure S17), while the final photoproduct obtained at 18.3 min was identified as the target active complex [Ru(biqbpy)(H2 O)2 ]2+ (Figure S14). Using the calibration curve previously obtained with this bis-aqua product, it was possible to quantitatively determine that after 24 h of green light irradiation of a 200 µg/mL RuBIS CPNs suspension (PBS buffer release), 1.1 µg/mL solution of the activated complex was obtained. 3.3. Cellular Uptake Measurements Skin non-melanoma A431 cells were incubated in the dark for 2 h with RuBIS CPNs, and the amount of Ru in the cells was quantified using ICP-MS. To better define the role of the nanostructuration on the internalization and phototherapy, we repeated these experiments with a related molecular complex [Ru(biqbpy)(N-methylimidazole)2 ](PF6 )2 (complex 2), which was especially synthesized as a reference model. Complex 2, a mononu- clear analogue of complex 1 coordinated with 2 axial methylimidazole ligands, was se- lected as a reference molecular complex for RuBIS CPNs due to the analogous ruthenium coordination sphere in both systems, which facilitates and makes more suitable the com- parative studies. Complex 2 was obtained upon the reaction of complex 1 with an excess of 1-methylimidazole (ratio 1:10) in EtOH under reflux and N2 atmosphere (for more information, see the Materials and Methods section). Remarkably, the time-dependent UV-Vis evolution of complex 2 under green light irradiation (Figure S18) is very similar to that previously observed for complex 1 (Figure S12) [14], validating its use for further biological studies. Nanomaterials 2021,11, Nanomaterials2021, 11,3089 x FOR PEER REVIEW 10 10 ofof1617 Figure5.5.(a) Figure (a)Time-dependent Time-dependentHPLCHPLCchromatogram chromatogramevolution evolutionunder underirradiation irradiationofofa aRuBIS RuBISCPNs CPNs colloidal suspension (200 µg/mL). (b) Plot of the time-dependent variation of relative UV absorption colloidal suspension (200 µg/mL). (b) Plot of the time-dependent variation of relative UV absorption ofofeach eachcomponent component(at (atgiven givenretention retentiontimes) times)upon uponirradiation. irradiation.Detection Detectionwavelength: wavelength:λλ==305305nm. nm. 3.3. As Cellular can beUptake seen Measurements in Figure 6, while complex 2 uptake (16 ng Ru/million cell) is com- parable to similar Skin molecular non-melanoma A431Rucells complexes previously were incubated reported in the [14], dark for 2 hRuBIS CPNs CPNs, with RuBIS were taken up by cells up to 11 times more (183 ng Ru/million cell). Such difference and the amount of Ru in the cells was quantified using ICP-MS. To better define the role was tenta- tively of theassigned to the different nanostructuration on the internalization internalizationmechanisms. To confirm and phototherapy, it, additional we repeated theseRuex- quantification studies were performed co-incubating RuBIS CPNs or periments with a related molecular complex [Ru(biqbpy)(N-methylimidazole)2](PF complex 2 with the6)2 endocytosis (complex 2),inhibitors which was NaN 3 , NH4 Cl, especially or Dynasore, synthesized as awhich are inhibitors reference of active model. Complex 2, uptake, a mono- endocytosis, and dynamin-dependent endocytosis, respectively. Indeed, nuclear analogue of complex 1 coordinated with 2 axial methylimidazole ligands, cellular uptake was of RuBIS CPNs was slightly decreased in the presence of dynasore (96.3 ng Ru/million selected as a reference molecular complex for RuBIS CPNs due to the analogous ruthe- cells), ammonium chloride (101 ng Ru/million cells), and to a lesser extent of sodium nium coordination sphere in both systems, which facilitates and makes more suitable the azide (138 ng Ru/million cells), compared to treatment with RuBIS CNPs alone (183 ng comparative studies. Complex 2 was obtained upon the reaction of complex 1 with an Ru/million cells). For the mononuclear complex 2, the opposite result was observed, with excess of 1-methylimidazole (ratio 1:10) in EtOH under reflux and N2 atmosphere (for a higher uptake in the presence of those inhibitors compared to treatment with 2 alone. more information, see the Materials and Methods section). Remarkably, the time-depend- Overall, these results suggested that endocytosis-related processes may be involved in ent UV-Vis evolution of complex 2 under green light irradiation (Figure S18) is very sim- cellular uptake of RuBIS CNPs, as reported previously for other nanoparticles [59], while ilar to that previously observed for complex 1 (Figure S12) [14], validating its use for fur- for complex 2, different mechanisms probably take place [60]. ther biological studies. As can be seen in Figure 6, while complex 2 uptake (16 ng Ru/million cell) is compa- rable to similar molecular Ru complexes previously reported [14], RuBIS CPNs were taken up by cells up to 11 times more (183 ng Ru/million cell). Such difference was tenta- tively assigned to the different internalization mechanisms. To confirm it, additional Ru quantification studies were performed co-incubating RuBIS CPNs or complex 2 with the of RuBIS CPNs was slightly decreased in the presence of dynasore (96.3 ng Ru/million cells), ammonium chloride (101 ng Ru/million cells), and to a lesser extent of sodium azide (138 ng Ru/million cells), compared to treatment with RuBIS CNPs alone (183 ng Ru/mil- lion cells). For the mononuclear complex 2, the opposite result was observed, with a higher uptake in the presence of those inhibitors compared to treatment with 2 alone. Overall, Nanomaterials 2021, 11, 3089 these results suggested that endocytosis-related processes may be involved in cellular up- 11 of 16 take of RuBIS CNPs, as reported previously for other nanoparticles [59], while for com- plex 2, different mechanisms probably take place [60]. Figure Figure6.6.Cellular uptake Cellular quantification uptake for Ru for quantification content Ru in A431 cells content treated in A431 withtreated cells complex 2 (19complex 2 with µg/mL) or RuBIS (19 µg/mL) CPNs (25 or RuBIS µg/mL) CPNs (25nanoparticles for 2 h in thefor µg/mL) nanoparticles presence 2 h inorthe absence of uptake presence in- or absence of up- hibitors: ammonium chloride, dynasore, or sodium azide endocytosis. take inhibitors: ammonium chloride, dynasore, or sodium azide endocytosis. 3.4. 3.4.(Photo)cytotoxicity (Photo)cytotoxicityStudies Studies RuBIS RuBISCPNs CPNs dispersions dispersions (from 0.7 ng/mL (from 0.7 ng/mLto 2.83toµg/mL based on 2.83 µg/mL Ru content) based or on Ru content) or complex 2 solutions (from 1.0 ng/mL to 5.0 ug/mL based on Ru content) in Opti-MEM complex 2 solutions (from 1.0 ng/mL to 5.0 ug/mL based on Ru content) in Opti-MEM medium (with minimum amounts of DMSO < 0.5% to fully dissolve complex 2) were medium (with minimum amounts of DMSO < 0.5% to fully dissolve complex 2) were seeded in human skin (A431) and lung cancer (A549) cell lines. As described in previous seeded[61], reports in human the cellsskin were(A431) seededand at t lung cancerat(A549) = 0, treated t = 24 h,cell lines. As irradiated described with in previous green light (λexc = 520 nm, 39.3 J/cm ) for 1 h at 48 h, and its cell viability was quantified at t = 96with reports [61], the cells 2 were seeded at t = 0, treated at t = 24 h, irradiated h green 2 ) for 1 h at 48 h, and its cell viability was quantified at using a standard light (λ exc = 520sulforhodamine B (SRB) assay. Half-effective growth inhibition concen- nm, 39.3 J/cm tration t = 96 h(EC 50 in µM) using values atsulforhodamine a standard t = 96 h are shownBin(SRB) Tableassay. 1, and Half-effective dose–response curvesgrowth areinhibition shown in Figure(EC concentration S19.50Cisplatin was alsoattested in µM) values t = 96under h arethe same in shown conditions Table 1,and andused as dose–response control. curves are shown in Figure S19. Cisplatin was also tested under the same conditions and usedTheas EC 50 values obtained for RuBIS CPNs in the dark (EC50,dark) of 11.88 µM and 9.10 control. µM decrease under irradiation (EC50,light) to 4.95 µM and 5.04 µM for A431 and A549 cells, respectively Table 1. Cytotoxicity of RuBIS CPNs and (Table 1). Therefore, complex 2 in A431 andthereA549 is a cancer remarkable difference cell lines in the in the dark ECunder and 50 values for green light 2 (λexc = 520 nm, 39.3 J/cm ) the RuBIS CPNs with and without irradiation, showing cell-dependent phototoxicity in- irradiation. dex (PI) values of 2.4 and 1.8 toward A431 and A549 cells, respectively. In both cases, the RuBISofCPNs PI values RuBIS CPNs are higher than Complex those of 2 the molecular complex 2 Cisplatin (1.7 and 1.0, Cell Light Type DoseJ/cm 2 EC respectively). 50 From these data,EC the 50 following considerations deserve EC 50to be mentioned. CI [a] PI [b] CI [a] PI [b] CI [a] PI [b] (µM) First, the EC50,dark of RuBIS CPNs (µM)was higher than that of complex 2, which was expected, (µM) as it is +0.46 possibly related to the significant +0.06 higher uptake. Second, the EC50,light+0.45 values of 0 11.9model complex 2 were 16.33 µM28.1 and 27.52 µM in A431 and A549 cells, 3.0respectively, which −n.a. −0.60 −0.41 A431 are up to 3–5 five times 2.4 higher than those found for RuBIS 1.7 CPNs, confirming the positive 1.1 +0.04 +0.55 +0.31 39.3 5.0 16.3 3.3 −0.04 −0.32 −0.28 +0.09 +1.16 +0.15 0 9.1 28.3 3.0 −0.08 −0.74 −0.15 A549 1.8 1.0 1.0 +0.02 +0.43 +0.17 39.3 5.0 27.5 3.0 −0.02 −0.37 −0.17 [a] Confidence interval, [b] photo indices. EC50 values are expressed in µM as half-maximal effective concentration (95% confidence interval are also given in µM). The EC50 values obtained for RuBIS CPNs in the dark (EC50,dark ) of 11.88 µM and 9.10 µM decrease under irradiation (EC50,light ) to 4.95 µM and 5.04 µM for A431 and A549 cells, respectively (Table 1). Therefore, there is a remarkable difference in the EC50 values for the RuBIS CPNs with and without irradiation, showing cell-dependent phototoxicity index (PI) values of 2.4 and 1.8 toward A431 and A549 cells, respectively. In both cases, the PI values of RuBIS CPNs are higher than those of the molecular complex 2 (1.7 and 1.0, respectively). From these data, the following considerations deserve to be mentioned. Nanomaterials 2021, 11, 3089 12 of 16 First, the EC50,dark of RuBIS CPNs was higher than that of complex 2, which was expected, as it is possibly related to the significant higher uptake. Second, the EC50,light values of model complex 2 were 16.33 µM and 27.52 µM in A431 and A549 cells, respectively, which are up to 3–5 five times higher than those found for RuBIS CPNs, confirming the positive nanostructuration effect on the enhancement of phototoxicity. These results imply that less Ru is required to photoinduce an efficient chemotherapeutic effect, minimizing the cells death for non-irradiated cells. Moreover, the obtained EC50,light values are similar to previously described monomeric complexes [14] but using almost a half-irradiation dose, which is also an additional advantage given the possible undesirable side effects that may appear from using a high irradiation doses. Last but not least, EC50,dark values for RuBIS CPNs are three to four times higher than those of cisplatin (3.01 and 3.04 µM), i.e., less toxic; while EC50,light values of RuBIS CPNs were close to those obtained for the photo-independent gold-standard cisplatin (EC50 = 3.01 µM and 3.04 µM in A431 and A549, respectively), which equate their effectiveness to drugs commercially used today. Cytotoxicity of the bis-imidazol (bis) and methylimidazole ligands are considered negligible, since a previous evaluation in our group using concentrations ranging from 0 to 100 µg/mL in different cell lines (Figure S20) corroborated their very low cytotoxic effect at the highest concentration assayed (100 µg/mL). 3.5. Singlet Oxygen Production To discard PDT as a possible origin of the photoactivity instead of PACT, the produc- tion of singlet oxygen (1 O2 ) upon green light irradiation of RuBIS CPNs was quantified. For this, the common method is to measure the near-infrared emission intensity of 1 O2 (1270 nm) in CD3 OD; though in this case and to mimic the cell culture conditions, the value of singlet oxygen quantum yield (Φ∆ ) was indirectly determined in Opti-MEM medium using a selective water-soluble 1 O2 probe (9,10-anthracenediyl-bis(methylene)-dimalonic acid, ABMDMA). In the dark, this dye absorbs light at 378 nm while in the presence of photo-generated 1 O2 , a less conjugated endoperoxide is formed, leading to a decrease in the absorbance at 378 nm [62]. The rose Bengal dye was used as reference, as it produces 1 O2 with a known quantum yield ϕ∆ = 0.76 [53] under green light irradiation. When RuBIS CPNs (25 µg/mL) was mixed with ABMDMA (100 µM) in Opti-MEM, no changes in the absorption spectra were observed with or without green light irradiation (λexc = 520 nm, see Figure 7), contrary to rose Bengal (Figure S21) [63]. The same study was performed Nanomaterials 2021, 11, x FOR PEER REVIEW 13 of 17 in the same way for complex 2. In both cases, the results excluded 1 O2 production, as expected for photosubstituted active ruthenium compounds. Figure7.7.The Figure Thenegligible negligible absorption absorption spectral spectral changes changes of ABMDMA of ABMDMA upon upon green green lightlight irradiation irradiation in thein the presence of (a) RuBIS CPNs (25 μg/mL), (b) complex 2 (19 μg/mL). The 1O2 generation is studied presence of (a) RuBIS CPNs (25 µg/mL), (b) complex 2 (19 µg/mL). The 1 O2 generation is studied in Opti-MEM medium. The arrows indicate the evolution of the spectra with time. in Opti-MEM medium. The arrows indicate the evolution of the spectra with time. 4. Conclusions We have successfully designed and synthetized light-sensitive coordination polymer nanoparticles (CPNs) based on the polymerization of a Ru(II) polypyridyl prodrug 1 with a photocleavable bis-imidazole linking ligand BIS. Precise control of the reaction condi- tions led to the reproducible synthesis of narrow size distribution (50 ± 12 nm) CPNs with Nanomaterials 2021, 11, 3089 13 of 16 4. Conclusions We have successfully designed and synthetized light-sensitive coordination polymer nanoparticles (CPNs) based on the polymerization of a Ru(II) polypyridyl prodrug 1 with a photocleavable bis-imidazole linking ligand BIS. Precise control of the reaction conditions led to the reproducible synthesis of narrow size distribution (50 ± 12 nm) CPNs with remarkable drug encapsulation yields well over those already described for other nanoen- capsulation systems. The photoactivation of the RuBIS CPNs showed controlled release of the anticancer Ru complex [Ru(biqbpy)(H2 O)2 ]2+ upon green (532 nm) irradiation, while they were stable in cell-growing medium in the dark, reducing the cell dead population and side effects in its inactivated form. Interestingly, the dose of light necessary to ob- tain enough cytotoxic complex from RuBIS CPNs in vitro (39.3 J/cm2 ) is notably lower compared to previous values published for similar green light photoactivated ruthenium systems (75 J/cm2 ) [14]. Moreover, in vitro studies demonstrated that RuBIS CNPs have an 11-fold increased uptake in comparison to related monomeric complexes thanks to the energy-dependent endocytosis uptake pathway triggered by the CNPs formulation. This fact determined a substantial increase in phototoxicity index in comparison with monomeric species and a light-selective cytotoxic effect close to the gold standard cisplatin. All in all, RuBIS CPNs demonstrates the potential of photoactivated CPNs for PACT anticancer treatments. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/nano11113089/s1, Figure S1: 1 H-NMR spectrum of complex 2 in (CD3 )2 SO, Figure S2: COSY spectrum of complex 2 in (CD3 )2 SO, Figure S3: 13 C-NMR spectrum of complex 2 in (CD3 )2 SO, Figure S4: Mass spectrum of complex 2, Figure S5: FTIR spectra of complex 1, BIS ligand and RuBIS CPNs, Figure S6: Time-dependent DLS measurements RuBIS CPNs, Figure S7: X-ray Diffractometry of BIS ligand, KPF6 , complex 1, and RuBIS CPNs, Figure S8: Calibration curve of the Ru for inductively coupled plasma mass spectrometry (ICP-MS), Figure S9a: 1 H NMR spectra of RuBIS CPNs, complex 1, CH2 FCN (internal reference) and BIS ligand, Figure S9b: 19 F NMR spectra of RuBIS CPNs and CH2 FCN (internal reference), Figure S10: (a) UV- vis absorption spectra of a PBS suspension of RuBIS CPNs in the dark over time. (b) Plot of absorbance at λabs = 315 and 439 nm of the RuBIS CPNs suspension in the dark. (c) DLS tracing results of the RuBIS CPNs suspension in PBS solution recorded at 0 min and 3 h 10 min, Figure S11: (a) Evolution of the UV-vis spectra of a PBS suspension of RuBIS CPNs, upon blue light irradiation (λex = 450 nm). (b) Plot of absorbance changes at λabs = 326 nm and 439 nm, Figure S12: Evolution of the UV-vis absorbance spectra of a solution of complex 1, upon 450 nm blue or 530 nm green light irradiation under argon. Reprinted with permission from Ref. [14]. Copyright 2016 the Royal Society of Chemistry. Figure S13: The whole chromatogram of fully activated drug [Ru(biqbpy)(H2 O)2 ]2+ converted from complex 1 at different concentrations, Figure S14: Mass spectrometry of the fractions at different retention times), Figure S15: The drug ([Ru(biqbpy)(H2 O)2 ]2+ ) calibration curve, Figure S16: The whole chromatogram of the drug release of RuBIS CPNs (0–35 min), Figure S17: Mass spectra of samples with retention time, Figure S18: UV-vis absorbance spectra of a complex 2 solution after green light irradiation, Figure S19: Cytotoxicity assays with and without green light (520 nm) irradiation, Figure S20: Cytotoxicity of Bis and 1-methylimidazole free ligands, Figure S21: Absorption spectral changes of ABMDMA in the dark (a) or following green light irradiation with the light of dose 39.3 J/cm2 , (b) in the presence of Rose Bengal (0.1 µM), Figure S22: Determination of required irradiation time for RuBIS CPNs and complex 2 activation. Author Contributions: J.Z. synthesized the materials, performed all the physicochemical characteri- zations. V.R. conducted in vitro studies and X.-Q.Z. conducted singlet oxygen generation studies. C.F. conducted preliminary studies and generated proof of concept results, D.R.-M. participated in the design of the experiments and discussion of the results. S.B. supervised the in vitro results and participated in the discussion results. C.R. and F.N. supervised the whole research work and coordinated the different studies. The manuscript was written through contributions of all authors. All authors have read and agreed to the published version of the manuscript. Nanomaterials 2021, 11, 3089 14 of 16 Funding: This work was supported by grant RTI2018-098027-B-C21 funded by MCIN/AEI/10.13039/ 501100011033 and by ERDF A way of making Europe. With the support from “Metalfármacos multi- funcionales para el diagnóstico y la terapia” with grant RED2018-102471-T funded by MCIN/AEI/ 10.13039/501100011033. The ICN2 is funded by the CERCA programme/Generalitat de Catalunya. The ICN2 is supported by the Severo Ochoa Centres of Excellence programme, grant SEV-2017-0706 funded by MCIN/AEI/10.13039/501100011033. J.D. Zhang thanks the BIST PhD Fellowship Pro- gramme (This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 754558). NWO is kindly acknowledged for financial support to SB via a VICI grant. COST is kindly acknowledged for stimulating scientific discussion and financial support via the Cost Action CA 17140 “Cancer nanomedicine from the bench to the bedside”. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is available on the request from the corresponding author. Acknowledgments: The authors thanks Jose Bolanos Cardet for the design of the Figure 1 and Graphical Abstract. Conflicts of Interest: The authors declare no conflict of interest. References 1. Kladnik, J.; Kljun, J.; Burmeister, H.; Ott, I.; Romero-Canelón, I.; Turel, I. Towards Identification of Essential Structural Elements of Organoruthenium(II)-Pyrithionato Complexes for Anticancer Activity. Chem. A Eur. J. 2019, 25, 14169–14182. [CrossRef] [PubMed] 2. Zhao, Z.; Gao, P.; You, Y.; Chen, T. Cancer-Targeting Functionalization of Selenium-Containing Ruthenium Conjugate with Tumor Microenvironment-Responsive Property to Enhance Theranostic Effects. Chem. A Eur. J. 2018, 24, 3289–3298. [CrossRef] 3. Hartinger, C.G.; Zorbas-Seifried, S.; Jakupec, M.A.; Kynast, B.; Zorbas, H.; Keppler, B.K. 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