Selective Photokilling of Human Pancreatic Cancer Cells Using Cetuximab-Targeted Mesoporous Silica Nanoparticles for Delivery of Zinc Phthalocyanine

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Article Selective Photokilling of Human Pancreatic Cancer Cells Using Cetuximab-Targeted Mesoporous Silica Nanoparticles for Delivery of Zinc Phthalocyanine

Özge Er 1 , Suleyman Gokhan Colak 2, Kasim Ocakoglu 3, Mine Ince 3, Roger Bresolí-Obach 4 , Margarita Mora 5, Maria Lluïsa Sagristá 5, Fatma Yurt 1,* and Santi Nonell 4,*

1 Department of Nuclear Applications, Institute of Nuclear Science, Ege University, Bornova, Izmir 35100, Turkey; [email protected] 2 Advanced Technology Research & Application Center, Mersin University, Ciftlikkoy Campus, Yenisehir, Mersin 33343, Turkey; [email protected] 3 Department of Energy Systems Engineering, Faculty of Technology, Tarsus University, Tarsus 33400, Turkey; [email protected] (K.O.); [email protected] (M.I.) 4 Institut Quimic de Sarria, Universitat Ramon Llull, Via Augusta 390, 08017 Barcelona, Spain; [email protected] 5 Departament de Bioquímica i Biomedicina Molecular, Facultat de Biologia, Universitat de Barcelona, Avinguda Diagonal 645, E-08028 Barcelona, Spain; [email protected] (M.M.); [email protected] (M.L.S.) * Correspondence: [email protected] (F.Y.); [email protected] (S.N.); Tel.: +90232-311-34-34 (F.Y.); +34-93-267-2000 (S.N.) Academic Editor: Michael R. Hamblin Received: 9 October 2018; Accepted: 23 October 2018; Published: 24 October 2018

Abstract: Background: Photodynamic therapy (PDT) is a non-invasive and innovative cancer therapy based on the photodynamic effect. In this study, we sought to determine the singlet oxygen production, intracellular uptake, and in vitro photodynamic therapy potential of Cetixumab-targeted, zinc(II) 2,3,9,10,16,17,23,24-octa(tert-butylphenoxy))phthalocyaninato(2-)-N29,N30,N31,N32 (ZnPcOBP)- loaded mesoporous silica nanoparticles against pancreatic cancer cells. Results: The quantum yield (Φ∆) value of ZnPcOBP was found to be 0.60 in toluene. In vitro cellular studies were performed to determine the dark- and phototoxicity of samples with various concentrations of ZnPcOBP by using pancreatic cells (AsPC-1, PANC-1 and MIA PaCa-2) and 20, 30, and 40 J/cm2 light ﬂuences. No dark toxicity was observed for any sample in any cell line. ZnPcOBP alone showed a modest photodynamic activity. However, when incorporated in silica nanoparticles, it showed a relatively high phototoxic effect, which was further enhanced by Cetuximab, a monoclonal antibody that targets the Epidermal Growth Factor Receptor (EGFR). The cell-line dependent photokilling observed correlates well with EGFR expression levels in these cells. Conclusions: Imidazole-capped Cetuximab-targeted mesoporous silica nanoparticles are excellent vehicles for the selective delivery of ZnPcOBP to pancreatic cancer cells expressing the EGFR receptor. The novel nanosystem appears to be a suitable agent for photodynamic therapy of pancreatic tumors.

Keywords: Zn(II) phthalocyanine; mesoporous silica nanoparticles; Cetuximab; singlet oxygen; photodynamic therapy

1. Introduction Pancreatic cancer is the 13th most common cancer and the 4–8th leading cause of cancer deaths worldwide. It occurs in the majority of cases with early locoregional spread and distant metastases [1,2]. The lack of symptoms in early stages causes late diagnoses, which detracts from the effectiveness

Molecules 2018, 23, 2749; doi:10.3390/molecules23112749 www.mdpi.com/journal/molecules Molecules 2018, 23, 2749 2 of 14 of treatments such as surgery, radiotherapy, or chemotherapy [1,3]. Before 2008, the only potential curative treatment offered to patients was a combination of surgery and adjuvant chemotherapy, with a five-year survival rate near to 40%. Targeted radionuclide therapies, using radiolabeled peptides capable of binding to receptors overexpressed by cancer cells or radiolabeled antibodies for tumor-specific antigens, and therapies directed against the Epidermal Growth Factor Receptor (EGFR) [4,5] have been proposed as viable alternatives to chemotherapy and external beam radiation therapies. Targeting agents for EGFR include tyrosine kinase inhibitors and monoclonal antibodies such as Cetuximab (C225). Photodynamic therapy (PDT) is a treatment modality that combines light, a drug acting as a photosensitizer (PS) and tissue oxygen, all innocuous for the cells by themselves, to eliminate tumors [6]. It can be used for the treatment of various ophthalmic, dermatologic, cardiovascular, and predominantly, oncologic diseases. The light-activated PSs create highly reactive oxygen species 1 such as singlet oxygen ( O2), which destroy cells in the tissue [7,8]. Phthalocyanines (Pcs) show strong potential as PSs owing to their strong absorption in the far visible region of the solar spectrum (between 640 and 700 nm), which matches the ‘therapeutic window’ (600–800 nm). Moreover, they are chemically, 1 photochemically, and thermally stable and present high efficiency of O2 generation [9–13]. However, many Pcs are hydrophobic and little soluble in aqueous medium, where they tend to aggregate, which 1 weakens their photophysical properties and O2 production ability [14]. The use of delivery vehicles such as nanoparticles (NPs), has emerged as a valuable option to overcome these limitations [15,16]. In addition, NPs have the capability to selectively accumulate in the malignant tissue, either passively, by the enhanced permeability and retention (EPR) effect [17], or actively, if their surface is decorated with ligands that can be selectively recognized by specific markers of the tumor cells [18,19]. Thus, these targeted drug delivery systems are able to modify the pharmacokinetics and biodistribution of their associated drugs. Indeed, nanomedicine is starting to make an impact in areas such as disease imaging diagnosis and drug delivery [20,21]. In recent years, the use of NPs in PDT has increased dramatically due to both the ability to deliver a large number of PS molecules to a single cell and the enhanced targeting potential offered by surface decoration with suitable ligands, which may significantly reduce the systemic toxicity associated with classical PDT therapy [21–23]. Although several nanomaterials have been explored for nanomedicine [16], silica nanoparticles, particularly of the mesoporous type (MSNPs), appear of great interest for PDT due to their easy preparation, wide range of sizes, biocompatibility, high drug loading capacity, internalization by a large number of cell lines, chemical inertness, large surface area, and easy surface decoration with ligands and solubilizing agents [24–26]. Of specific interest for this work, MSNPs decorated with EGFR-targeting monoclonal antibody Cetuximab have been used for the delivery of doxorubicin and gefitinib to PC9-DR cells and tumors [27]. Thus, the aim of this study has been to assess the potential of MSNPs decorated with Cetuximab for the photodynamic killing of pancreatic cancer cells.

2. Results

2.1. Synthesis of Zinc(II) 2,3,9,10,16,17,23,24-octa(tert-butylphenoxy))phthalocyaninato(2-)-N29,N30,N31,N32 (ZnPcOBP) ZnPcOBP was synthesized in 23% yield by means of the cyclotetramerization of 4,5-di(p-tert- butylphenoxy)phthalonitrile in the presence of ZnCl2 in dimethylaminoethanol (DMAE; Scheme1). The spectroscopic data are in agreement with published data [28]. Molecules 2018, 23, 2749 3 of 14 Molecules 2018, 23, x FOR PEER REVIEW 3 of 15 Molecules 2018, 23, x FOR PEER REVIEW 3 of 15

O O O O

N N N N N NC O ZnCl2, DMAE O N O NC O ZnCl DMAE O O 2, N Zn N Reflux, overnight N Zn N O NC O Reflux, overnight O O NC O O N N N N N N

O O O O

SchemeScheme 1. Synthesis1. Synthesis of Zinc (II) 2,3,9,10,16,17,23,24-octa(of Zinc (II) 2,3,9,10,16,17,23,24-octa(tert-butylphenoxy)) phthalocyaninato(2-)-tert-butylphenoxy)) Scheme 1. Synthesis of Zinc (II) 2,3,9,10,16,17,23,24-octa(tert-butylphenoxy)) N29phthalocyaninato(2-)-N,N30,N31,N32 (ZnPcOBP).29, N30, N31,N32 (ZnPcOBP). phthalocyaninato(2-)-N29, N30, N31,N32 (ZnPcOBP). 2.2.2.2. Preparation Preparation of of Nanoparticles Nanoparticles 2.2. Preparation of Nanoparticles TheThe preparation preparation of of nanoparticles nanoparticles waswas carriedcarried out in in five ﬁve steps steps (Scheme (Scheme 2):2): (i) (i) preparation preparation of ofthe the The preparation of nanoparticles was carried out in five steps (Scheme 2): (i) preparation of the complexcomplex between between ZnPcOBP ZnPcOBP and 3-(2-imidazol-1-yl)propyltriethoxysilaneand 3-(2-imidazol-1-yl)propyltriethoxysilane (ITES-ZnPc); (ITES-ZnPc); (ii) preparation (ii) complex between ZnPcOBP and 3-(2-imidazol-1-yl)propyltriethoxysilane (ITES-ZnPc); (ii) ofpreparation mesoporous of silica mesoporous nanoparticles silica with nanoparticles entrapped ZnPcOBPwith entrapped (MSNP2); ZnPcOBP (iii) modiﬁcation (MSNP2); of(iii) the preparation of mesoporous silica nanoparticles with entrapped ZnPcOBP (MSNP2); (iii) nanoparticlemodification surface of the withnanoparticle mercaptopropyl surface with groups mercaptopropyl (MSNP3); (iv)groups anchoring (MSNP3); of polyethyleneglycol(iv) anchoring of modification of the nanoparticle surface with mercaptopropyl groups (MSNP3); (iv) anchoring of polyethyleneglycol chains (PEG; MSNP4); (v) anchoring of cetuximab (MSNP5). A chainspolyethyleneglycol (PEG; MSNP4); chains (v) anchoring (PEG; ofMSNP4); cetuximab (v) (MSNP5). anchoring A phthalocyanine-freeof cetuximab (MSNP5). nanoparticle A phthalocyanine-free nanoparticle analog to MSNP5 was prepared as control (MSNP1). analogphthalocyanine-free to MSNP5 was nanoparticle prepared as analog control to (MSNP1). MSNP5 was prepared as control (MSNP1).

Scheme 2. Preparation of the nanoparticles. SchemeScheme 2.2. PreparationPreparation of the nanoparticles. nanoparticles.

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Molecules 2018, 23, x FOR PEER REVIEW 4 of 15 2.3. Characterization of Nanoparticles 2.3. Characterization of Nanoparticles The size and zeta potential of the nanoparticles were measured by dynamic light scattering The size and zeta potential of the nanoparticles were measured by dynamic light scattering (DLS). The results are shown in Table1. ZnPcOBP-containing nanoparticles were larger than (DLS). The results are shown in Table 1. ZnPcOBP-containing nanoparticles were larger than phthalocyanine-free nanoparticles due to the grafting of ITES, the increase in size and concomitant phthalocyanine-free nanoparticles due to the grafting of ITES, the increase in size and concomitant changes in the zeta potential after each preparation step being a good indication that the sequential changes in the zeta potential after each preparation step being a good indication that the sequential surface modiﬁcations had been successfully accomplished. surface modifications had been successfully accomplished. Table 1. Size and zeta potential of MSNP1, MSNP3, MSNP4 and MSNP5 nanoparticles. Table 1. Size and zeta potential of MSNP1, MSNP3, MSNP4 and MSNP5 nanoparticles. Title 1 MSNP1 MSNP3 MSNP4 MSNP5 Title 1 MSNP1 MSNP3 MSNP4 MSNP5 Size (nm) 126 ± 8 262 ± 15 285 ± 27 303 ± 26 Size (nm) 126 ± 8 262 ± 15 285 ± 27 303 ± 26 Zeta Potential (mV) −14 ± 4 −27 ± 1 −17 ± 5 −9 ± 7 Zeta Potential (mV) −14 ± 4 −27 ± 1 −17 ± 5 −9 ± 7

FieldField emission-scanningemission-scanning electronelectron microscopymicroscopy (FE-SEM)(FE-SEM) andand transmissiontransmission electronelectron microscopymicroscopy (TEM)(TEM) werewere used used to to obtain obtain images images of of the the nanoparticles nanoparticles (Figures (Figures S1 S1 and and S2 respectively).S2 respectively). The The size size of the of MSNP3the MSNP3 nanoparticles nanoparticles was determinedwas determined to be to 60 be± 1060 nm± 10 by nm analysis by analysis of the of FE-SEM the FE-SEM images. images. In the In TEM the images,TEM images, the porous the structureporous ofstructure MSNP3 of can MSNP3 be clearly can seen be (Figure clearly S2). seen In addition,(Figure energy-dispersiveS2). In addition, X-rayenergy-dispersive spectroscopy X-ray (EDS, spectroscopy Figure S3) conﬁrmed (EDS, Figure the presence S3) confirmed of Si, O andthe S,presence whereby of the Si, detectedO and S, S elementwhereby represents the detected -SH S groupselement on represents the surface -SH of groups the nanoparticle. on the surface of the nanoparticle. TheThe IRIR spectra spectra of of ZnPcOBP, ZnPcOBP, ITES, ITES, the complexthe comple ITES-ZnPc,x ITES-ZnPc, and the and nanoparticles the nanoparticles MSNP3, MSNP3, MSNP4, andMSNP4, MSNP5 and are MSNP5 shown are in shown Figure S4.in Figure Comparison S4. Comparison of the spectra of the for spectra MSNP4 for and MSNP4 MSNP5 and show MSNP5 that cetuximabshow that cetuximab is attached is to attached the nanoparticles to the nanopartic in MSNP5les in as MSNP5 indicated as byindicated the appearance by the appearance of two new of −1 bandstwo new (1641 bands and 1534(1641 cm and), 1534 assigned cm−1), to assigned the normal to modesthe normal of vibration modes of of amide vibration I and of amide amide II ofI and the antibodyamide II of backbone, the antibody respectively backbone, [29 ].respectively [29]. UV-VISUV-VIS spectraspectra ofof ZnPcOBPZnPcOBP inin toluene,toluene, acetonitrile,acetonitrile, and methanol are shown in Figure 11a.a. ZnPcOBPZnPcOBP isis inin monomeric monomeric form form in in toluene, toluene, as as deduced deduced from from the the presence presence of twoof two sharp sharp peaks peaks at 370 at nm370 andnm and 680 nm.680 nm. However, However, it is slightlyit is slightly aggregated aggregated in acetonitrile in acetonitrile and extensively and extensively aggregated aggregated in the in more the polarmore solventpolar solvent methanol, methanol, as indicated as indicated by the by decrease the decrease in absorbance in absorbance and broadening and broadening of the bands.of the Thebands. UV-VIS The UV-VIS absorbance absorbance spectra ofspectra MSNP3 of andMSNP3 MSNP4 and (1MSNP4 mg) dispersed (1 mg) dispersed in methanol in aremethanol shown are in Figureshown1 b.in Figure It is worth 1b. It noting is worth that noting when that the suspensionswhen the suspensions were centrifuged, were centrifuged, no ZnPcOBP no couldZnPcOBP be foundcould inbe the found supernatant. in the supernatant. The similarity The with similarity the spectrum with ofthe ZnPcOBP spectrum in methanolof ZnPcOBP indicates in methanol that the phthalocyanineindicates that the is phthalocyanine extensively aggregated is extensively in the NPs.aggregated in the NPs.

FigureFigure 1.1. ((aa)) AbsorbanceAbsorbance spectrumspectrum ofof ZnPcOBPZnPcOBP inin toluenetoluene (blue),(blue), acetonitrileacetonitrile (red),(red), andand methanolmethanol (black);(black); ((bb)) AbsorbanceAbsorbance spectraspectra ofof MSNP3MSNP3 (blue)(blue) andand MSNP4MSNP4 (red)(red) inin methanol.methanol.

TheThe amountamount ofof ZnPcOBPZnPcOBP inin thethe nanoparticlesnanoparticles was determined by extracting it with THF, measuringmeasuring its absorbance absorbance at at 680 680 nm, nm, and and interpolatin interpolatingg in a in calibration a calibration curve curve (ZnPcOBP (ZnPcOBP in THF in in THF the µ µ inrange the range0.1–1 0.1–1μM; FigureM; Figure S5). S5).Thus, Thus, it was it was observed observed that that 1 mg 1 mg MSNP3 MSNP3 contains contains 10.4 10.4 μg ZnPcOBP (Figure S6), 1 mg MSNP4 contains 6.2 μg ZnPcOBP (Figure S7), and 1 mg MSNP5 contains 3.8 μg

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(FigureZnPcOBP S6), (Figure 1 mg S8), MSNP4 consistent contains with 6.2 theµg increase ZnPcOBP of the (Figure nanoparticle S7), and mass 1 mg upon MSNP5 sequential contains addition 3.8 µg ZnPcOBPof surface (Figurecomponents. S8), consistent with the increase of the nanoparticle mass upon sequential addition of surface components. 2.4. Singlet Oxygen Production 2.4. Singlet Oxygen Production When exposed to light, many PSs kill cancer cells by producing singlet oxygen. We therefore assessedWhen the exposed ability toof light,ZnPcOBP many to PSs photosensitize kill cancer cells the byproduction producing of singletthis reactive oxygen. oxygen We therefore species. assessedThus, small the amounts ability of of ZnPcOBPa concentrated to photosensitize stock solution the of productionZnPcOBP inof toluene this reactive were diluted oxygen into species. 2 mL Thus,toluene small and amountsthe time-dependent of a concentrated change stock of phosphorescence solution of ZnPcOBP intensity in toluene upon werepulsed diluted laser excitation into 2 mL toluenewas recorded and the at time-dependent 1275 nm (where change singlet of oxygen phosphorescence emits) and intensity at 1110 nm upon (where pulsed the laser triplet excitation ZnPcOBP was recordedemits). Unequivocal at 1275 nm (where1O2 production singlet oxygen was emits)thus demonstrated and at 1110 nm by (where the observation the triplet ZnPcOBPof a long-lived emits). 1 Unequivocalrise-and decayO signal2 production at 1275 wasnm, thuswhile demonstrated at 1110 nm only by thea short-lived observation decay of a long-livedsignal could rise-and be observed decay signal(Figure at 2a). 1275 nm, while at 1110 nm only a short-lived decay signal could be observed (Figure2a).

Figure 2.2. Time-resolvedTime-resolvedphosphorescence phosphorescence intensity intensity 1275 1275 nm nm (black) (black) and and 1110 1110 nm nm (green) (green) upon upon 355 355 nm pulsednm pulsed laser laser excitation excitation of (a) ZnPcOBPof (a) ZnPcOBP in toluene; in toluene; (b) MSNP3 (b) in MSNP3 methanol; in (methanol;c) MSNP4 in(c) methanol.MSNP4 in methanol. The quantum yield of singlet oxygen production (Φ∆; Figure S9) was determined by comparing the 1275The quantum nm phosphorescence yield of singlet intensity oxygen production of ZnPcOBP (ΦΔ with; Figure that S9) of was a determined reference photosensitiser, by comparing 1theH-phenalen-1-one 1275 nm phosphorescence (PN), for which intensityΦ∆ = of 0.92 ZnPcOB [30–32].P Wewith found that thatof a thereferenceΦ∆ value photosensitiser, of ZnPcOBP in1H toluene-phenalen-1-one is 0.60, in (PN), good for agreement which Ф∆ with = 0.92 the [30–32]. value of We 0.73 found reported that the by MareeΦΔ value and of Nyokong ZnPcOBP [28 in] intoluene chloroform. is 0.60, in good agreement with the value of 0.73 reported by Maree and Nyokong [28] in chloroform.The production of singlet oxygen by the nanoparticles was likewise investigated in methanol suspensions.The production For this of study, singlet 1 mg oxygen of MSNP3 by the and nanoparticles 1 mg of MSNP4 was likewise were dispersed investigated in 1 mL in methanol andsuspensions. the phosphorescence For this study, was 1 recordedmg of MSNP3 at 1275 and and 1 mg 1110 of nm. MSNP4 As shown were indispersed Figure2 b,c,in 1 the mL two methanol traces areand indistinguishable, the phosphorescence which was indicates recorded that at 1275 MSNP3 and and 1110 MSNP4 nm. As do shown not release in Figure any 2b,c, detectable the two amount traces ofare singlet indistinguishable, oxygen. As an which additional indicates control, that the MSNP3 experiments and MSNP4 were repeated do not in release the presence any detectable of sodium azideamount (NaN of singlet3), a well-known oxygen. As singlet an additional oxygen quenchercontrol, the [33 experiments]. Again, the were signals repeated did not in change, the presence which confirmsof sodium that azide production (NaN3), ofa singletwell-known oxygen singlet by the oxygen nanoparticles quench iser negligible [33]. Again, (Figure the S10).signals did not change, which confirms that production of singlet oxygen by the nanoparticles is negligible (Figure 2.5. Release of ZnPcOBP from the Nanoparticles S10). We observed that ZnPcOBP could be quantitatively extracted from the nanoparticles by using non-polar2.5. Release solvents, of ZnPcOBP such from as THF, the Nanoparticles or coordinating solvents, such as pyridine (Figure S11). We therefore hypothesizedWe observed that that proteins, ZnPcOBP widely could available be quantitatively in biological extracted media, might from bethe able nanoparticles to extract ZnPcOBP by using fromnon-polar the nanoparticles solvents, such facilitating as THF, theiror coordinating delivery to the solvents, cancer cells.such Toas testpyridine this hypothesis, (Figure S11). 2 mg We of µ µ MSNP4therefore or hypothesized MSNP5 and that 250 proteins,L of bovine widely serum availabl albumine in biological (BSA) 150 media,M were might dispersed be able in to 2 extract mL of phosphate-bufferedZnPcOBP from the nanoparticles saline (PBS; pH facilitating 7.4) and the their mixtures deliver allowedy to the tocancer equilibrate cells. To overnight test this under hypothesis, gentle magnetic2 mg of MSNP4 stirring. or The MSNP5 suspensions and 250 were μL of then bovine centrifuged serum andalbumin the fluorescence (BSA) 150 μ ofM the were supernatants dispersed wasin 2 recorded.mL of phosphate-buffered Nanoparticle suspensions saline (PBS; without pH BSA7.4) wereand the used mixtures as controls. allowed The characteristic to equilibrate fluorescence overnight under gentle magnetic stirring. The suspensions were then centrifuged and the fluorescence of the supernatants was recorded. Nanoparticle suspensions without BSA were used as controls. The

Molecules 2018, 23, x FOR PEER REVIEW 6 of 15 Molecules 2018, 23, 2749 6 of 14 characteristic fluorescence bands of ZnPcOBP were remarkably more intense in the samples containing BSA (Figure 3a,b), confirming that ZnPcOBP can be released from the nanoparticles in bands of ZnPcOBP were remarkably more intense in the samples containing BSA (Figure3a,b), the presence of proteins. confirming that ZnPcOBP can be released from the nanoparticles in the presence of proteins. Since cell incubation media usually contain 10% fetal bovine (or calf) serum, we investigated the Since cell incubation media usually contain 10% fetal bovine (or calf) serum, we investigated release of ZnPcOBP from the nanoparticles in the presence of human serum (HS). Figure 3c,d shows the release of ZnPcOBP from the nanoparticles in the presence of human serum (HS). Figure3c,d that the release, measured by fluorescence of the supernatant after centrifugation, is negligible under shows that the release, measured by fluorescence of the supernatant after centrifugation, is negligible typical incubation conditions (PBS containing 10% human serum incubated for up to 48 h). under typical incubation conditions (PBS containing 10% human serum incubated for up to 48 h). Increasing the concentration of human serum and the incubation time led to concomitantly higher Increasing the concentration of human serum and the incubation time led to concomitantly higher release efficiencies. release efficiencies.

Figure 3. Fluorescence spectra of the supernatant after centrifugation of (a) MSNP4 and (b) MSNP5 Figure 3. Fluorescence spectra of the supernatant after centrifugation of (a) MSNP4 and (b) MSNP5 nanoparticle suspensions incubated overnight in the presence (red) and absence (black) of BSA. nanoparticle suspensions incubated overnight in the presence (red) and absence (black) of BSA. Inﬂuence of human serum (HS) content on the release of ZnPcOBP from (c) MSNP4 and (d) MSNP5 Influence of human serum (HS) content on the release of ZnPcOBP from (c) MSNP4 and (d) MSNP5 nanoparticles as a function of the incubation time in PBS-HS mixtures. nanoparticles as a function of the incubation time in PBS-HS mixtures. 2.6. Cellular Uptake of MSNP4 and MSNP5 2.6. Cellular Uptake of MSNP4 and MSNP5 We studied the cellular uptake for MSNP4 and MSNP5 nanoparticles on the pancreatic tumor cell linesWe PANC-1, studied AsPC-1,the cellular and uptake MIA PaCa-2. for MSNP4 AsPC-1 and and MSNP5 PANC-1 nanoparticles cells express on more the EGFRpancreatic than tumor MIA PaCa-2cell lines cells PANC-1, [34]. Thus, AsPC-1, appropriate and MIA amounts PaCa-2. of AsPC-1 the nanoparticles and PANC-1 were cells added express to the more cells EGFR to deliver than 1 MIAµM ZnPcOBP PaCa-2 cells and [34]. the extent Thus, of appropriate uptake was amounts assessed of at fourthe nanoparticles incubation times were (1 added h, 3 h, 6to h the and cells 24 h). to Thedeliver results 1 μ areM summarizedZnPcOBP and in the Figure extent4. of uptake was assessed at four incubation times (1 h, 3 h, 6 h andA 24 consistent h). The results increase are insummarized ZnPcOBP ﬂuorescence in Figure 4. per microgram of cellular protein was observed upon increasingA consistent the increase incubation in ZnPcOBP time for fluorescence all cell lines. per For microgram any given of cell cellula line,r nanoparticleprotein was observed uptake wasupon higher increasing for MSNP5 the incubation than for time MSNP4, for all consistent cell lines. For with any the given presence cell line, and nanoparticle absence, respectively, uptake was ofhigher Cetuximab for MSNP5 on their than surface. for MSNP4, The enhancement consistent waswith cell the dependent, presence and namely absence, 10%, respectively, 23%, and 46% of higherCetuximab in MIA-PaCa-2, on their surface. PANC-1, The and enhancement AsPC-1 cells, was respectively, cell dependent, in agreement namely 10%, with 23%, the differentand 46% expressionhigher in levelsMIA-PaCa-2, of EGFR PANC-1, in these cells and [ 34AsPC-1]. In absolute cells, respectively, terms, the highest in agreement cellular uptakewith the of MSNP5different nanoparticlesexpression levels was observedof EGFR in in thes PANC-1e cells cells, [34]. followed In absolute by terms, AsPC-1 th ande highest MIA PaCa-2cellular cells. uptake of MSNP5 nanoparticles was observed in PANC-1 cells, followed by AsPC-1 and MIA PaCa-2 cells.

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Figure 4. Cellular uptake kinetics of MSNP4 and MSNP5 nanoparticles on PANC-1, AsPC-1 and MIA FigurePaCa-2 4. cells. Cellular uptake kinetics of MSNP4 and MSNP5 nanoparticles on PANC-1, AsPC-1 and MIA PaCa-2 cells. 2.7. Cell Photocytotoxicity Studies 2.7. CellIn vitroPhotocytotoxicitycell photocytotoxicity Studies studies were carried out by using PANC-1, AsPC-1, and MIA PaCa-2 cellIn lines. vitro Water-insoluble cell photocytotoxicity ZnPcOBP studies and MSNP3 were carried nanoparticles out by using were firstPANC-1, dissolved AsPC-1, or suspended, and MIA PaCa-2respectively, cell lines. in dimethylacetamide Water-insoluble ZnPcOBP (DMA) and and then MSNP3 added nanoparticles to the incubation were medium first dissolved of the cells. or suspended,MSNP1, MSNP4, respectively, and MSNP5 in dimethylacetamide nanoparticles were (DMA delivered) and then suspended added to in the PBS. incubation Cells were medium incubated of thein thecells. dark MSNP1, for 24 MSNP4, h with and various MSNP5 concentrations nanoparticles of were free ZnPcOBPdelivered orsuspended the nanoparticles in PBS. Cells and were then incubatedexposedto in variousthe dark fluences for 24 h ofwith LED various red light concentrations or kept in the of free dark ZnPcOBP for control or purposes.the nanoparticles Finally, and cell thenviability exposed was to assessed various 24h fluences after the of treatment.LED red light Figure or kept5 shows in the the dark photocytotoxicity for control purposes. results afterFinally, the celldifferent viability treatments. was assessed 24h after the treatment. Figure 5 shows the photocytotoxicity results after the differentExperiments treatments. carried out by adding the PS or NPs in DMA revealed that ZnPcOBP alone had only a modestExperiments photodynamic carried out activity. by adding Moreover, the PS DMA or NPs exerted in DMA a substantial revealed darkthat ZnPcOBP toxicity by alone itself had and onlyenhanced a modest the dark-photodynamic and phototoxicity activity. of Moreover, the MSNP3 DM nanoparticles,A exerted a substantial preventing anydark sound toxicity comparison by itself andwith enhanced the results the of dark- the other and nanosystemsphototoxicity (Figureof the S12).MSNP3 On nanoparticles, the contrary, thepreventing dark toxicity any sound of the comparisonnanosystems with delivered the results in PBS of the was other negligible nanosystem (Figures 5(Figurea,e,i). S12). On the contrary, the dark toxicity of theMSNP1 nanosystems nanoparticles, delivered whichin PBS containwas negligible no ZnPcOBP, (Figure showed5a,e,i). almost no dark toxicity for the PANC-1MSNP1 and nanoparticles, AsPC-1 cells, butwhich cell contain viability no decreased ZnPcOBP, in ashowed concentration-dependent almost no dark toxicity manner for for the the PANC-1MIA-PaCa-2 and AsPC-1 cells down cells, to but 60% cell at theviability maximum decreased concentration in a concentration-dependent (Figure5b,f,j). The dark manner toxicity for of the MIA-PaCa-2other nanosystems cells down (MSNP4 to 60% and at MSNP5) the maximum was very concen similartration to that (Figure of MSNP1, 5b,f,j). rulingThe dark out toxicity any important of the othercontribution nanosystems of the (MSNP4 ZnPcOBP and or Cetuximab.MSNP5) was PANC-1 very similar and AsPC-1 to that cells of viabilityMSNP1, inruling the dark, out afterany importanttheir incubation contribution with ZnPcOBP-loaded-MSNP4 of the ZnPcOBP or Cetuximab. (coated with PANC-1 PEG; Figureand AsPC-15c,g,k) cells and -MSNP5viability (coated in the dark,with PEGafter andtheir Cetuximab; incubation Figure with5 d,h,l)ZnPcOBP-loaded nanoparticles,-MSNP4 was near (coated to 80% with for thePEG; higher Figure photosensitizer 5c,g,k) and -MSNP5dose, the (coated MIAPaCa-2 with PEG cells and again Cetuximab; being more Figure sensitive 5d,h,l) with nanoparticles, a cell viability was ofnear 70% to at80% the for higher the higherphotosensitizer photosensitizer dose. dose, the MIA PaCa-2 cells again being more sensitive with a cell viability of 70% atUpon the higher irradiation, photosensitizer a decrease dose. in cell viability was observed that was concentration and light- fluenceUpon dependent. irradiation, Under a decrease the same in experimental cell viability conditions,was observed cell viabilitythat was was concentration lower when and cells light- were fluenceincubated dependent. with MSNP5 Under nanoparticles the same experimental than with MSNP4 conditions, nanoparticles. cell viability Thus, was at lower 5.0 µM when ZnPcOBP cells 2 wereand aincubated light fluence with of 40MSNP5 J/cm nanoparticles, the cell viability than was with 35%, MSNP4 55%, andnanoparticles. 39% for the Thus, PANC-1, at 5.0 AsPC-1, μM ZnPcOBPand MIA PaCa-2and a light cells fluence incubated of with40 J/cm MSNP4,2, the cell respectively, viability whilewas 35%, the corresponding55%, and 39% valuesfor the for PANC-1, MSNP5 AsPC-1, and MIA PaCa-2 cells incubated with MSNP4, respectively, while the corresponding values

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for MSNP5 were 6.2%, 12.5%, and 17.5%. Again, these results are in agreement with the cell uptake were 6.2%, 12.5%, and 17.5%. Again, these results are in agreement with the cell uptake and EGFR and EGFR expression level in these cells. expression level in these cells.

Figure 5. ASPC-1 (a–d), PANC-1 (e–h), and MIA PaCa-2 (i–l) cell viability (expressed as percentages) afterFigure incubation 5. ASPC-1 with: (a–d), PBS PANC-1 (a,e,i), (e MSNP1–h), and (MIAb,f,j), PaCa-2 MSNP4 (i–l ()c ,cellg,k )viability or MSNP5 (expressed (d,h,l) as nanoparticles percentages) suspendedafter incubation in PBS, with: at various PBS (a ZnPcOBP,e,i), MSNP1 concentrations (b,f,j), MSNP4 and ( lightc,g,k) ﬂuences or MSNP5 (20, ( 30,d,h, andl) nanoparticles 40 J/cm2). 2 Insuspended the panels in (a ,PBS,e,i), equivalentat various ZnPcOBP amounts ofconcentrat PBS to thoseions neededand light to fluences reach the (20, indicated 30, and concentrations40 J/cm ). In the ofpanels ZnPcOBP (a,e,i were), equivalent added inamounts the wells of toPBS assess to those the effectneeded of to medium reach the dilution indicated with concentrations PBS. ZnPcOBP of concentrationsZnPcOBP were are added 0.1 µM in (black), the wells 0.5 µ Mto (red),assess 1.0 theµM effect (blue), of andmedium 5 µM (green).dilution Cellswith incubatedPBS. ZnPcOBP with completeconcentrations growth are medium, 0.1 μM to (black), which 100%0.5 μM viability (red), 1.0 was μM assigned, (blue), wereand 5 used μM as(green). controls Cells to calculateincubated thewith cell complete viability ofgrowth the cells medium, treated to at which the different 100% viab conditions.ility was Data assigned, are the were mean used± SD asfrom controls three to independentcalculate the experiments. cell viability of the cells treated at the different conditions. Data are the mean ± SD from three independent experiments. 3. Discussion 3. Discussion The results above highlight the potential of MSNPs coated with Cetuximab for the photodynamic killingThe of pancreaticresults above cancer highlight cells. Mesoporous the potential silica of nanoparticles MSNPs coated have with been engineeredCetuximab forfor thethe selectivephotodynamic delivery killing of ZnPcOBP of pancreatic to the cells. cancer The sizecells. of theMesoporous nanoparticles silica (260–300 nanoparticles nm) is adequate have been for photodynamicengineered for applications the selective and delivery their slightly-negative of ZnPcOBP to the zeta cells. potential The size precludes of the theirnanoparticles aggregation (260–300 into largernm) is structures. adequate for photodynamic applications and their slightly-negative zeta potential precludes theirThe aggregation coordination into ability larger of structures. its central Zn cation allows reversible binding of the phthalocyanine to nanoparticlesThe coordination with imidazoles ability of grafted its central onto Zn their cation surface. allows This reversible kind of bindingbinding facilitatesof the phthalocyanine the release to nanoparticles with imidazoles grafted onto their surface. This kind of binding facilitates the

Molecules 2018, 23, 2749 9 of 14 into the cancer cells. Indeed, it was found that ZnPcOBP could be extracted from the nanoparticles using suitable solvents, as well as BSA and serum proteins. While in principle this might lead to a premature release in the presence of blood proteins, it should not be difficult to address this issue at the pharmacology stage, modifying the nanoparticle structure and binding affinity. 1 Regarding its photosensitizing properties, ZnPcOBP is an excellent O2 PS with a quantum yield 1 of Φ∆ = 0.6 in the monomeric form. However, it does not produce O2 when bound to the nanoparticles because it is extensively aggregated (see Figure1) and it is well known that aggregation suppresses 1 the ability of phthalocyanines to produce O2 [35]. ZnPcOBP aggregation may prove advantageous to avoid unwanted photo-induced damage to other components in its pharmaceutical formulations. When delivered in DMA solutions, ZnPcOBP showed only a small photodynamic activity against cancer cells, in contrast with the results for its nanoparticle-bound formulations. Indeed, MSNP3 nanoparticles show quite high phototoxic effects against AsPC-1, PANC-1, and MIA PaCa-2 cell cultures; however, this is probably due to the DMA solvent needed to solubilize them, which makes them also quite toxic in the dark. Their phototoxicity might be due, at least in part, to extraction of ZnPcOBP by DMA and/or cell membrane disruption by DMA favoring nanoparticle internalization. In contrast, MSNP4 and MSNP5 are dispersible in PBS and show phototoxicity on AsPC-1, PANC-1, and MIA PaCa-2 cells, MSNP5 nanoparticles being superior to MSNP4. This is due to the presence of the Cetuximab antibody on their surface that favors cell recognition and receptor-mediated cell internalization, which, in turn, allows the release of ZnPcOBP within the cells. In vitro photodynamic results are consistent with cellular uptake and largely reflect the different levels of EGFR expression on the three cell lines. Therefore, the results presented clearly suggest that nanoparticles coated with PEG and cetuximab (MSNP5) could be an efficient vehicle for delivery of the photosensitizer ZnPcOBP to tumoral pancreatic cells with high EGFR expression in a targeted fashion.

4. Materials and Methods

4.1. Materials Triethoxy-3-(2-imidazolin-1-yl)propylsilane (ITES), hexadecyl trimethyl ammonium chloride (CTAC), triethylamine (TEA), tetraethyl orthosilicate (TEOS), (3-mercaptopropyl) trimethoxysilane (MPS), Mal-PEG-COOH, 1-ethyl-3 (3-dimethylaminopropyl) carbodimide (EDC) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Cetuximab (Erlotinib) was supplied from Merck Serono (Darmstadt, Germany). The chemicals used in the in vitro studies were supplied from CLS-Cell Line Service. The other chemicals were purchased from Merck (Darmstadt, Germany). 4,5-Di(p-tert-butylphenoxy)phthalonitrile was synthesized as described previously [36].

4.2. Synthesis and Derivatitzation of Mesoporous Silica Nanoparticles

4.2.1. Synthesis of Zinc (II) 2,3,9,10,16,17,23,24-octa(tert-butylphenoxy))phthalocyaninato (2-)-N29,N30,N31,N32 (ZnPcOBP)

A mixture of 4,5-di(p-tert-butylphenoxy)phthalonitrile (300 mg, 0.7 mmol) and ZnCl2 (53 mg, 0.28 mmol) in dimethylaminoethanol (DMAE; 5 mL) was heated at reﬂux overnight with stirring under argon atmosphere. After cooling to room temperature, the solvent was removed and the residue was washed with a MeOH/H2O (5:1) mixture. The crude product was puriﬁed by column chromatography on silica gel, (DCM/THF, 10:1) as eluent to yield ZnPcOBP (80 mg, 0.04 mmol) as a green solid. Yield: 23%. 1 H-NMR (400 MHz, CDCl3): δ (ppm) = 8.93–8.45 (m, 8H), 7.40 (d, 4H, J =8 Hz), 7.3–7.2 (m, 12H), 7.1–7.0 (m 12H), 6.8 (d, 4H, J = 8 Hz), 1.32–1.29 (m, 72H). IR (ATR): ν (cm−1) = 3647, 2955, 1719, 1602, 1506, 1487, 1446, 1362, 1291, 1178, 890, 720. UV-Vis (CHCl3): λmax (log ε) = 676 (1.46), 646 (2.05), 608 (2.23), 358 (4.51). MS (MALDI): m/z: 1763.205 [M+]. Molecules 2018, 23, 2749 10 of 14

4.2.2. Preparation of ITES-ZnPc Complex The ITES-ZnPc complex was prepared by combining of ZnPcOBP and ITES at the molar ratio of 1:10 following the study of Durgun et al. [37]. ZnPcOBP (0.011 mmol) was dissolved in 2 mL of absolute ethanol under magnetic stirring. Then, 0.11 mmol ITES was added to the mixer and stirred for 24 h in the dark and at room temperature. After 24 h, the ITES-ZnPc core was collected and used without further puriﬁcation.

4.2.3. Synthesis of Mesoporous Silica Nanoparticles and Encapsulation of ZnPcOBP (MSNP2) The mesoporous silica nanoparticles were synthesized based on the method used by Chen et al. [38]. A quantity of 2.0 g of CTAC (6.2 mmol) and 20 mg of triethylamine (TEA; 0.2 mmol) were dissolved in 30 mL of bidistilled water and mixed at room temperature for 1 h. Then, the previously prepared ITES-ZnPc complex was added to the mixture and stirred for an additional hour. Afterwards, 2.0 mL of TEOS (9.0 mmol) was added rapidly and the resulting mixture was stirred at 95 ◦C for 1 h. At the end of the reaction, the mixture was allowed to cool and centrifuged to collect (10 min, 3000 rpm, room temperature). The collected product (MSNP2) was then washed with water and ethanol to remove residual reagents. Then, the product was extracted three times with 1 wt% NaCl solution in methanol for 3 h to remove the CTAC.

4.2.4. Synthesis of MSNP3 MSNP2 nanoparticles were functionalized with -SH groups as described in ref. [38]. All of the synthesized MSNP2 nanoparticles were dispersed in 20 mL absolute ethanol and 2.0 mL of (3-mercaptopropyl)trimethoxysilane (MPS; 11 mmol) were added. The mixture was closed and kept in the dark under stirring for 12–24 h at 86–90 ◦C. Afterwards, the reaction mixture was cooled down and centrifuged (10 min, 3000 rpm, room temperature). The obtained nanoparticles (MSNP3) were washed several times with ethanol to remove the residual MPS.

4.2.5. Synthesis of MSNP4 MSNP3 nanoparticles were PEGylated with Mal-PEG-COOH chain as described by Karra et al. [39]. MSNP3 nanoparticles were dispersed in water (pH 6.5–7) in a round bottom flask. Then the same amount of Mal-PEG-COOH was added to the flask (1:1 wt ratio of MSNP3:Mal-PEG-COOH) and stirred for 24 h at room temperature. Later, MSNP4 were collected from the flask, centrifuged and washed with water for 3 times (10 min, 3000 rpm, room temperature).

4.2.6. Synthesis of MSNP5 Conjugation of Cetuximab to the surface of the MSNP4 nanoparticles was performed according to Wang et al. [19]. MSNP4 nanoparticles were dispersed in puriﬁed water and the pH was adjusted to 4.8. Then EDC (5:2 wt ratio of MSNP4:EDC) was added and the mixture was left under stirring at room temperature for 30 min. Afterwards, the carboxyl-activated MSNP4 nanoparticles were centrifuged and washed twice with PBS. Next, the activated NPs and Cetuximab (5:2 wt ratio of activated NPs:Cetuximab) were allowed to react overnight in PBS at room temperature. Finally, MSNP5 were centrifuged and washed with PBS.

4.2.7. Synthesis of MSNP1 MSNP1 nanoparticles were prepared following the same procedure as for MSNP5 without adding ITES-ZnPc.

4.2.8. Nanoparticle Characterization The size and morphology of the synthetized NPs were characterized using: (i) ﬁeld emission scanning electron microscopy (FE-SEM; Zeiss/Supra 55, Carl Zeiss AG, Oberkochen, Germany), Molecules 2018, 23, 2749 11 of 14

(ii) transmission electron microscopy (TEM; JEOL JEM-2100 (UHR), Jeol Ltd., Tokyo, Japan) and (iii) dynamic light scattering (DLS; Zetasizer-Malvern Nano ZS90, Malvern Instruments Ltd., Worcestershire, UK) techniques. ATR-infrared spectra of the NPs were recorded in a Spectrum Two FTIR-ATR (Perkin Elmer, Waltham, MA, USA).

4.3. Spectroscopic Techniques UV-Vis absorption spectra of the samples in various solvents were recorded with a Varian Cary 6000i spectrophotometer (Palo Alto, CA, USA). Fluorescence spectra of the samples were measured with a Fluoromax-4 spectrofluorometer (Horiba Jobin-Ybon, Edison, NJ, USA). 1 Production of O2 was studied by time-resolved near-infrared phosphorescence using a setup described in detail elsewhere [40,41]. Briefly, a pulsed Nd:YAG laser (FTSS355-Q, Crystal Laser, Berlin, Germany) working at 355 nm (third harmonic; 0.5 µJ per pulse; 1 kHz repetition rate) was used for sample excitation. A 1064 nm rugate notch filter and an uncoated SKG-5 filter were placed at the exit port of the laser to remove any residual component of its fundamental emission in the near-infrared region. The luminescence exiting from the sample was filtered by a 1100 nm long-pass filter and a narrow bandpass filter at 1275 nm. A thermoelectric-cooled near-infrared 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). 1 The kinetic parameters governing the production and decay of O2 were determined by fitting the model below to the time-resolved phosphorescence intensity at 1275 nm (Equation (1)):

S(t) = S(0) × τ∆/(τ∆ − τT) × [exp(−t/τ∆) − exp(−t/τT)] (1)

1 where τ∆ and τT are O2 and triplet ZnPcOBP lifetimes, and the pre-exponential factor S(0) is an 1 instrumental quantity proportional to the O2 production quantum yield (Φ∆). The Φ∆ value for ZnPcOBP was determined by comparison of its S(0) value to that of an optically-matched solution of the reference PS phenalenone in toluene, for which Φ∆ = 0.92 [30–32].

4.4. Cellular Techniques

4.4.1. Cell Lines and Culture Conditions Cell culture studies were carried out using AsPC-1 (human pancreatic adenocarcinoma), PANC-1 (human pancreas/duct epithelioid carcinoma), and MIA PaCa-2 (human pancreas carcinoma) cell lines. AsPC-1 cells were cultured in RPMI 1640 containing 10% heat-inactivated FBS, 1% L-glutamine, and 1% penicillin-streptomycin. PANC-1 and MIA PaCa-2 cells were cultured in DMEM containing 10% heat-inactivated FBS, 1% L-glutamine, and 1% penicillin-streptomycin. All the cells were maintained ◦ at 37 C in an incubator containing 5% CO2.

4.4.2. Cellular uptake Assays A quantity of 1.7 × 104 cells was added to 96 wells plate and incubated at 37 ◦C for 24 h to achieve an 80% confluence. Afterwards, the culture medium was removed and 1 µM photosensitizer (alone or adsorbed onto NPs) with fresh medium was added. After the corresponding incubation times, the compounds were removed, the wells were washed 3 times with PBS. Then 100 µL of SDS (4%) were added and incubated 30 min more at 37 ◦C. Then 100 µL DMA was added and incubated for 30 min more. Afterwards, the plates were shaken and ZnPcOBP fluorescence was read in a microplate reader (λexc 620 nm; λem 675 nm). The BCA protein kit was used for protein correlation after reading the ZnPcOBP fluorescence [42]. Thus, 150 µL from each well was transferred to a clear bottom 96 well plate, 150 µL of the BCA working reagent was added to each well, the plates were shaken for 30 s on a plate shaker, and they were finally incubated at 37 ◦C for 1 h. After incubation, the plates were cooled to room temperature and the Molecules 2018, 23, 2749 12 of 14 absorbance of each well was measured at 562 nm with a microplate reader. The amount of protein was calculated by using the standard curve of BSA. Then the fluorescence of the wells was divided with correlated BSA concentration. Therefore, the cellular uptake of compounds was determined as ZnPcOBP fluorescence/protein amount (µg).

4.4.3. In Vitro Dark- and Phototoxicity Assays A quantity of 1.7 × 104 cells was added to 96 wells plate and incubated at 37 ◦C for 24 h to achieve 80% conﬂuence. Afterwards, the culture medium was removed and the previously-dissolved ZnPcOBP or suspended NPs or the equivalent amounts of DMA or PBS were added. After 24 h of incubation, the compounds were removed, the wells were washed 3 times with PBS and then fresh medium was added onto the cells. Dark controls were placed directly into the 37 ◦C incubator for 24 h, whilst light-treated plates were irradiated with 20, 30, and 40 J/cm2. For photodynamic treatments, 2 the red light from an LED Par 64 Short V2 lamp (λem = 633 ± 9 nm; irradiance: 9.0 mW/cm ; Showtec, Kerkrade, The Netherlands) was used. After irradiation, the plates were placed into a 37 ◦C incubator for 24 h. Finally, the medium onto the cells was removed and the dark- and phototoxicity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay [43].

Supplementary Materials: Supplementary Materials are available online. Figure S1. SEM images of MSNP3 (a), MSNP4 (b) and MSNP5 (c). Figure S2. TEM images of MSNP3 (a), MSNP4 (b) and MSNP5 (c). Figure S3. EDS analysis graph of MSNP3 nanoparticles. Figure S4. Infrared spectra of ZnPcOBP (a), ITES (b), ITES-ZnPc (c), MSNP3 (d), MSNP4 (e) and MSNP5 (f). Figure S5. Top: UV-Vis absorption spectrum of 0.10–1.00 µM ZnPcOBP in tetrahydrofuran. Bottom: Calibration curve obtained from the absorbance data presented above at 680 nm. Figure S6. UV-Vis (a) and fluorescence (b; λexc = 620 nm) spectrum of 0.1–1 µM ZnPcOBP and 1 mg of MSNP3 in tetrahydrofuran. Figure S7. UV-Vis (a) and fluorescence (b; λexc = 620 nm) spectrum of 0.1–1 µM ZnPcOBP and 1 mg of MSNP4 in tetrahydrofuran. Figure S8. UV-Vis (a) and fluorescence (b; λexc = 620 nm) spectrum of 0.1–1 µM ZnPcOBP and 1 mg of MSNP5 in tetrahydrofuran. Figure S9. Singlet oxygen phosphorescence kinetic traces in toluene for optically-matched solutions of ZnPcOBP (green line) and phenalenone (PN; black line). λobs = 1275 nm. Figure S10. Singlet oxygen phosphorescence kinetic traces for MSNP3 (left) and MSNP4 (right) in the absence (black line) and presence (25 mM; green line) of NaN3 in methanol. λobs = 1275 nm. Figure S11. Right: One milliliter of MSNP-3 (left Eppendorf tube) and MSNP-4 (right Eppendorf tube) centrifuged for 10 min at 3000 rpm in pyridine. Left: UV-Vis spectra of the previous supernatants (black and red lines for MSNP-3 and MSNP-4 respectively). Figure S12. ASPC-1 (a–c), PANC-1 (d–f) and MIA PaCa-2 (g–i) cell viability (expressed in percentage) after incubation with: DMA (a,d,g) or ZnPcOBP (dissolved in DMA; b,e,h) and MSNP3 (suspended in DMA; c,f,i), at various ZnPcOBP concentrations and light fluences (20, 30 and 40 J/cm2). In the panels (a,d,g), equivalent amounts of DMA to those needed to reach the indicated concentrations of ZnPcOBP were added in the wells to assess the effect of medium dilution with DMA. ZnPcOBP concentrations are 0.1 µM (black), 0.5 µM (red), 1.0 µM (blue), and 5 µM (green). Cells incubated with complete growth medium, to which 100% viability was assigned, were used as controls to calculate the cell viability of the cells treated at the different conditions. Data are the mean ± SD from three independent experiments. Author Contributions: O.E. performed the experiments. S.G.C. synthesized ZnPcOBP. K.O. and M.I. conceived and supervised the synthesis of ZnPcOBP and preparation of nanoparticles. R.B.-O. conducted the singlet oxygen experiments. M.M. and M.L.S. designed, supervised and interpreted the cell culture studies. F.Y. and S.N. conceived, supervised and interpreted all the study. Funding: This research was funded by Ministerio de Economía y Competitividad (grant number CTQ2016-78454-C2-1-R), the Scientific and Technological Research Council of Turkey (TUBITAK; grant number 2214/A), Ege University, Scientific Research Project (BAP; grant Number: 16 NBE 001), and the European Social Funds and the SUR del DEC de la Generalitat de Catalunya (grant number 2017 FI_B2 00140). Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are available from the authors.

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