Phthalocyanines Functionalized with Aminobenzoic Acids and Their GO-Based Composites

Journal of C Carbon Research

Article Synthesis, Spectroscopic Characterization and Photoactivity of Zr(IV) Phthalocyanines Functionalized with Aminobenzoic Acids and Their GO-Based Composites

Leili Tahershamsi 1,* , Yuriy Gerasymchuk 1, Anna Wedzynska 1, Maciej Ptak 1, Iryna Tretyakova 2 and Anna Lukowiak 1

1 Institute of Low Temperature and Structure Research, PAS, ul. Okolna 2, 50-422 Wroclaw, Poland; [email protected] (Y.G.); [email protected] (A.W.); [email protected] (M.P.); [email protected] (A.L.) 2 Institute of General and Inorganic Chemistry, Palladina ave. 32/34, 03142 Kyiv, Ukraine; [email protected] * Correspondence: [email protected]

Received: 16 October 2019; Accepted: 17 December 2019; Published: 22 December 2019

Abstract: Two complexes of bis(aminobenzoato)zirconium(IV) phthalocyanine and their graphite oxide-based composites were synthesized and characterized in respect of their photochemical properties. Structures of phthalocyanines were confirmed by Mass and infrared spectroscopies. The absorption and photoluminescence spectra were investigated to show various behavior of the complexes in different media (dimethyl sulfoxide and saline). Optical technique (monitoring variation of absorption spectra of diphenylisobenzofuran used as an indicator) was used to prove the generation of reactive oxygen species (ROS) by under light irradiation in the range of the first biological window. The photoactivity of the materials was compared and discussed in terms of their potential ability to be used in biomedical applications, for example, as photosensitizers in photodynamic therapy.

Keywords: graphite oxide (GO); phthalocyanine complexes; ROS generation; photoluminescence spectroscopy; photodynamic therapy

1. Introduction Due to different abilities of phthalocyanines (Pcs), they have emerged huge interest in catalysis [1], semiconductors, photoconductors [2–4] and optical recording materials [5]. Despite their proficiency in the mentioned area, they are capable of use in medicine as photocytotoxic reagents [6]. Photodynamic therapy (PDT) is a clinically approved procedure for curing cancerous cells, which involves using photosensitizing reagents with the aid of an appropriate wavelength of visible or near infrared (NIR) light [7,8]. 1 Reactive oxygen species (ROS), such as O2,O2−•, OH• and H2O2, are responsible for cytotoxic effect which kills injured or microbial cells by a combinatorial effect of light [9,10]. Phthalocyanines are one of the most promising candidate in PDT; their role as photosensitizers and their activity in the red–NIR range (the first biological window) and high emission coefficients are not negligible [11–13]. Thus, their photochemical and photobiological properties have been widely reported in the literature [14–16]. Generally, Pcs and MPc complexes (M—Metal) are not soluble in water or dimethyl sulfoxide (DMSO). To use Pcs in pharmacology, good solubility of Pcs in water is preferred [17,18]. For this aim, metallophthalocyanines must be functionalized with hydrophilic organic/inorganic substituents

C 2020, 6, 1; doi:10.3390/c6010001 www.mdpi.com/journal/carbon C 2020, 6, 1 2 of 13

in lateral position, or small inorganic counter-ions (Cl− or OH−) or carboxylate groups should be substituted to metal cation of Pc complex in more or less axial position [17]. In our approach, the water solubility of the complexes is achieved by supplemental axial organic ligands attached to the central metal of the complex [19–21]. The choice of metal cation (Zr4+) was dictated by the fact that, in zirconium (or hafnium) phthalocyanine complexes, the coordination number of metal is usually 6. After the replacement of simple axial ligands, that is, Cl− and OH− by organic ligands, the coordination number of the metal increases to 8. Thus, when the complex is formed, metal has 2 additional coordination sites where at least 2 solvent molecules (water, DMSO, dimethylformamide (DMF), etc.) can be bonded. In addition, solubility may be significantly affected by hydrophilic groups present in axial ligands. These two factors cause that the zirconium complexes are relatively well soluble in water and DMSO [18]. Graphene family materials, such as graphene oxide and reduced graphene oxide, have been utilized in a large number of activities in biology field [22–24]. Recent studies showed that, for example, graphene oxide and its reduced forms have antibacterial properties towards Gram-positive, for example, Staphylococcus aureus and Gram-negative, for example, Escherichia coli, bacteria [25,26]. Antibacterial effect of graphene materials could be used to fabricate antimicrobial and biofilm resistant surfaces, for example, polymeric films for biomedical devices [27,28], antibacterial fabric materials [29] and graphene oxide-based membrane for water treatment [30–32]. There are different hypothesis for antibacterial effects of graphene materials. One is driven by spontaneous piercing of the membrane by nanoflakes of carboneous/graphene structures [33]. There is an interaction between basal hydrophobic region of graphene to hydrophobic inner region of plasma of membrane. Inducing cell damage in bacteria by extracting phospholipid molecules from the outer cell membrane [34] and direct oxidation of cellular components by graphene oxide [35] are the other pathways for integration of graphene oxide with bacterial cell membranes. Oxidative stress has another role to deactivate or kill bacteria which might be induced by generation of reactive oxygen species in the presence of graphene structures [35]. Embedding Pcs into graphene or similar nano supports might enhance their photo-physicochemical properties [36]. Macromolecules, such as Pc templates, have tendency to agglomerate in aqueous and biological media, which influence their absorption and emission properties. Due to a dye binding to a graphene carrier, agglomeration of molecules might be significantly reduced. Furthermore, transferring the dye into biological targets such as cells and tissues would be easier and less toxic to biological organisms while they are linked to a carrier [37]. In our previous research, new photosensitive nanomaterial with antibacterial photoactivity were introduced [38], where Pcs derivative was used together with graphene oxide and silver nanoparticles in a composite to enhance the antimicrobial effect. In this paper we reported synthesis and photochemical characterization of two new zirconium(IV) phthalocyanine derivatives and their graphite oxide-based composites. The absorbance of ZrPcs with aminobenzoate ligands in dimethyl sulfoxide and saline were compared. Absorption spectroscopy was used to determine dependence of absorbance on Pc concentration and singlet oxygen generation under light irradiation for both Pcs and graphite oxide (GO) composites. Photoluminescent properties were also investigated.

2. Materials and Methods

2.1. Materials Syntheses

2.1.1. Pc Complexes For axial substitution of zirconium (IV) phthalocyanine complex with 4-aminobenzoic acid (PABA) and 3,5-diaminobenzoic acid (DABA), a method described earlier was used [37,39]. Generally, in the ﬁrst step, dichlorozirconium (IV) phthalocyanine was obtained by template synthesis from phthaliimide (Alfa Aesar, Karlsruhe, Germany) and ZrCl4 (ABCR, Karlsruhe, Germany) in 2-methylnaphtlalene (Alfa Aesar) with heating. ZrPcCl2 was used as a precursor for a next step, where it reacted with 4-aminobenzoic acid (Alfa Aesar) or 3,5-diaminobenzoic acid (Alfa Aesar) in DMSO (Alfa Aesar) during C 2020, 6, x 4 of 13

3. Results

3.1. Characterization of Pc Complexes and Composites Two different zirconium (IV) phthalocyanines with mono and di-aminobenzoic functionalities axially substituted to the central metal ion and their GO composites were studied in this article (Figure 1). Graphite oxide flakes with dimensions of a few microns and a thickness of several dozen C 2020, 6, 1 3 of 13 of carbon layers (TEM and SEM images of derived materials are presented in Figure S1 in the Supplementary Materials) and with different oxide groups were used to link functionalized phthalocyanines.heating under reﬂux To forconfirm about 6the h. Thestructures, procedure FTIR is schematicallyand mass spectroscopy shown in Figure measurements1. The complexes were performed.of bis (4-aminobenzoato) Afterwards, the ZrPc optical and properties bis (3,5-diaminobenzoato) of these systems ZrPcwere werestudied further and singlet abbreviated oxygen as generationbis(PABA)ZrPc was examined. and bis(DABA)ZrPc, respectively.

4 HCl

H2N NH2

O O

O O O

OH Zr

Cl Cl N N H2N N N N N N Zr N + OR O H2N NH N 2 N N N N N N H2N N OH

O O H N 2 NH2 O O NH2

N N N N N N N N

FigureFigure 1. Synthesis ofof PcMClPcMCl22 andand bisbis(L) (L) ZrPc ZrPc complexes, complexes, where where zirconium zirconium was was used used as metalas metal ion ion (M) (M)and and 4-aminobenzoic 4-aminobenzoic acid acid (PABA) (PABA) or 3,5-diaminobenzoic or 3,5-diaminobenzoic acid acid (DABA) (DABA) were were used used as ligands as ligands (L). (L).

2.1.2.FTIR Pc Composites spectra of bis (PABA) ZrPc and bis (DABA) ZrPc and their composites, bis (PABA) ZrPc/GO and bisHighly (DABA) oxidized ZrPc/GO, graphite display oxide expected (GO) was features prepared (Figure according 2), forto example, the modified bands Brodie at 3464, method 3354 cm [40−1] (Figureand composite 2a) are attributed materials mainly were preparedto amine accordinggroups of ligands to a previous (Table S1, report Supplementary [9]. GO powder Materials). (0.5 Theg) was broad suspended contour at by 3500–3100 ultrasonication cm−1 is (900related W, to 22 OH kHz groups sonicator) on the in GO 5 mL surface of dimethylformamide (Figure 2b,d). The broad(Alfa Aesar).peaks observed Then, about in the 4 mgregion of bis of (L)3500–3100 ZrPc, where cm−1 both L = PABA in complexes/DABAcorrespondingly, and composites, wasare related added toto hydrogen the reaction bonding vessel. of ReactionNH2 groups mixture (Figure was 2a,c). placed Thein narrower an ice bath bands and observed put on between a magnetic 2841 stirrer. and 3081Dicyclohexylcarbodiimide cm−1 are assigned to (DCC,stretching 0.06 vibrations g) (ABCR) of was CH added groups. to the Strong mixture bands and in the the reaction 1516–1689 was left cm in−1 regionthe ice are bath assigned on the magnetic mainly to stirrer stretching for 12 vibrations h. After completion of carbonyl of groups the reaction, (C=O) biscoupled (L) ZrPc-modified to vibrations ofGO aromatic materials rings were and separated bending vibrations by centrifuge of amine and washed groups in 3 timesbis (PABA) with DMFZrPc and bis twice (DABA) with warmZrPc, respectively.ethanol to remove Bending unreacted C–O dyevibrations molecules. are Compositesassigned to were bands abbreviated observed as in bis(PABA)ZrPc the 1207–1288/GO cm and−1. Characteristicbis(DABA)ZrPc stretching/GO consecutively. vibrations of CNC groups are ascribed to bands observed in the 1207–1458 cm−1 region, while their bending components contribute to many bands in the 505–736 cm−1 and 843– 2.2. Instrumentation A spectrophotometer FLS980 (Edinburgh Instruments, Livingston, UK) with a xenon lamp was used for photoluminescence measurements. The spectra were recorded with the respective lamp and detector corrections. Absorption spectra of Pc solutions and Pc/GO suspensions were measured with a spectrophotometer Agilent CARY 5000 UV-Vis-NIR (Thermo Fisher Scientific, Walham, MA, USA). 1 Polycrystalline IR spectra in the mid-IR region (400–4000 cm− ) were measured using a Nicolet iS50 C 2020, 6, 1 4 of 13

1 FT-IR spectrometer (Thermo Fisher Scientiﬁc). The spectral resolution was set to 2 cm− . A Bruker apex ultra with electrospray ion injector (ESI-MS) (Bruker Optics Inc, Billerica, MA, USA) was used for mass spectroscopy measurements.

2.3. Materials Characterization Structure of complexes were conﬁrmed by ESI-MS analysis and IR spectroscopy. Mass spectra of compounds were recorded in DMF solutions. For FTIR spectra measurements samples were prepared as KBr pellets. For other optical measurements, stock solutions of ZrPcs in concentration of 0.5 mM were prepared by dissolving the compounds in dimethyl sulfoxide whereas the concentration of composites was about 18 mg/mL. Absorption spectra in the cases of two complexes of phthalocyanines, bis (PABA) ZrPc and bis (DABA) ZrPc, were recorded upon addition of 5 to 45 µL of ZrPc solution to the cuvette containing 2 mL DMSO and 5 to 125 µL of ZrPc to the cuvette containing 2 mL NaClaq (9 mg/mL) (the Beer-Lambert experiment, where absorbance versus dye concentration is plotted). 1 Generation of O2 upon irradiation was shown by an indirect chemical method, using 1 1,3-diphenylisobenzofuran (DPBF) as a probe [41]. DPBF reacts with O2 irreversibly, leading to a decrease in the absorption band at around 420 nm. The solution containing DPBF in DMSO (20 mg/mL) was prepared on the day of the analysis and stored in the dark. DPBF solution (14 µL) was added to a solution of the ZrPc complex (20 µL of solution in 2 mL DMSO) (to obtain absorbance level of a measured phthalocyanine solution in Q region around 1) or ZrPc composite (30 µL of suspension in 2 mL DMSO), which were then irradiated (from 10 s up to maximum 240 s) by red/NIR lamp (Philips, 100 W) which served as a light source that was located 30 cm far from a cuvette. Beer-Lambert graph for bis (PABA) ZrPc was plotted at 683 nm or 695 nm at which the absorption bands maxima were registered in DMSO and saline, respectively. For bis (DABA) ZrPc, graph data was registered at 690 nm and 700 nm in DMSO and saline, respectively. Photoluminescence and photoluminescence excitation spectra of phthalocyanine complexes were recorded in DMSO at 4 10 5 M concentration. × − 3. Results

3.1. Characterization of Pc Complexes and Composites Two different zirconium (IV) phthalocyanines with mono and di-aminobenzoic functionalities axially substituted to the central metal ion and their GO composites were studied in this article (Figure1). Graphite oxide flakes with dimensions of a few microns and a thickness of several dozen of carbon layers (TEM and SEM images of derived materials are presented in Figure S1 in the Supplementary Materials) and with different oxide groups were used to link functionalized phthalocyanines. To confirm the structures, FTIR and mass spectroscopy measurements were performed. Afterwards, the optical properties of these systems were studied and singlet oxygen generation was examined. FTIR spectra of bis (PABA) ZrPc and bis (DABA) ZrPc and their composites, bis (PABA) ZrPc/GO 1 and bis (DABA) ZrPc/GO, display expected features (Figure2), for example, bands at 3464, 3354 cm − (Figure2a) are attributed mainly to amine groups of ligands (Table S1, Supplementary Materials). 1 The broad contour at 3500–3100 cm− is related to OH groups on the GO surface (Figure2b,d). The broad 1 peaks observed in the region of 3500–3100 cm− both in complexes and composites, are related to hydrogen bonding of NH2 groups (Figure2a,c). The narrower bands observed between 2841 and 1 1 3081 cm− are assigned to stretching vibrations of CH groups. Strong bands in the 1516–1689 cm− region are assigned mainly to stretching vibrations of carbonyl groups (C=O) coupled to vibrations of aromatic rings and bending vibrations of amine groups in bis (PABA) ZrPc and bis (DABA) ZrPc, respectively. 1 Bending C–O vibrations are assigned to bands observed in the 1207–1288 cm− . Characteristic 1 stretching vibrations of CNC groups are ascribed to bands observed in the 1207–1458 cm− region, 1 1 while their bending components contribute to many bands in the 505–736 cm− and 843–904 cm− regions. The bending in-plane vibrations of CH groups are related to many bands located between 946 1 1 and 1386 cm− , while out-of-plane aromatic CH are observed at lower wavenumbers (417–904 cm− ). C 2020, 6, x 5 of 13

904 cm−1 regions. The bending in-plane vibrations of CH groups are related to many bands located between 946 and 1386 cm−1, while out-of-plane aromatic CH are observed at lower wavenumbers (417–904 cm−1). The characteristic stretching vibrations of rings in Pc are assigned to bands between 1102 and 1600 cm−1. The in- and out-of-plane bending vibrations of rings contribute to bands observed in the 505–1088 cm−1 and 417–830 cm−1, respectively [42] (Figure 2a). The characteristic bands of GO are clearly visible at 1068, 1612 and 1724 cm−1 for bis (PABA) ZrPc/GO and at 983, 1071, 1612 and 1725 cm−1 for bis (DABA) ZrPc/GO [43]. By coupling reaction between phthalocyanine and GO, new linkages (amide bonds; Pc–NH– CCO–GO)2020, 6, 1 are formed in the system. Therefore, new peaks related to amide bonds are expected in5 ofthe 13 FTIR spectra of composites [44]. Three distinct differences can be found in FTIR spectra of composites that confirm the amide bond formation in the composites: (i) decrease in intensity of bands1 The characteristic stretching vibrations of rings in Pc are assigned to bands between 1102 and 1600 cm− . corresponding to NH2 stretching vibrations (Figure 2a,c); (ii) appearance of one narrow peak at 33251 The in- and out-of-plane bending vibrations of rings contribute to bands observed in the 505–1088 cm− cm−1 in bis (PABA)1 ZrPc/GO and 3326 cm−1 in (bis (DABA) ZrPc/GO spectra (Figure 2b,d); (iii) and and 417–830 cm− , respectively [42] (Figure2a). The characteristic bands of GO are clearly visible at the increase of intensity1 and shifts of bands corresponding to the stretching vibrations1 of carbonyl 1068, 1612 and 1724 cm− for bis (PABA) ZrPc/GO and at 983, 1071, 1612 and 1725 cm− for bis (DABA) −1 ZrPc(C=O)/GO groups [43]. of amides at 1626 cm in both composites (Figure 2a,c).

a) bis(PABA)ZrPc c) bis(DABA)ZrPc Absorbance (arb. u.) Absorbance (arb. u.) (arb. Absorbance

d) bis(DABA)ZrPc/GO b) bis(PABA)ZrPcGO

3500 3000 2500 2000 1500 1000 500 3500 3000 2500 2000 1500 1000 500 Wavenumber (cm-1) Wavenumber (cm-1)

Figure 2. Fourier-transform infrared (FTIR) (FTIR) spectra spectra of of (a) (a) bis bis (PABA) (PABA) ZrPc, (b (b)) bis bis (PABA) ZrPc/GO, ZrPc/GO, (c) bisbis (DABA)(DABA) ZrPcZrPc andand (d)(d) bisbis (DABA)(DABA) ZrPcZrPc/GO./GO.

ByMass coupling spectroscopy reaction analysis between confirmed phthalocyanine the structure and of GO,Pc complexes. new linkages In ESI-MS (amide spectra bonds; of Pc–NH–CO–GO)phthalocyanine complexes are formed (Figure in the S2, system. Supplementa Therefore,ry Materials), new peaks peaks related with tothe amide molecular bonds mass are expectedmore than in exact the FTIR mass spectra of complex of composites could [be44 ].seen Three due distinct to one di fforerences two molecules can be found of insolvent FTIR spectra(DMF) ofextracoordinated composites that to confirm metal atom the amide of phthalocyanine bond formation complex in the composites:as presented (i) for decrease bis (PABA) in intensity ZrPc ofin bandsFigure corresponding3. Coordinated tosolvent NH2 stretchingmolecules are vibrations bonded (Figure more strongly2a,c); (ii) with appearance metal atom of onethan narrow axial ligand. peak 1 1 atMoreover, 3325 cm −as init is bis shown (PABA) in Figure ZrPc/GO 3, the and structure 3326 cm −breaksin (bis in (DABA)ionization ZrPc chamber/GO spectra of the spectrometer. (Figure2b,d); (iii)Fragmentation and the increase starts with of intensity removal and of amine shifts offunctionalities bands corresponding from the ligand to the followed stretching by vibrations detachment of 1 carbonylof phenone (C =groupsO) groups from of the amides ZrPc atstructure. 1626 cm− Inin this both step, composites two molecules (Figure of2a,c). DMF are bonded to the structureMass which spectroscopy will be removed analysis later confirmed during theionization structure process. of Pc In complexes. the case of Inbis ESI-MS (DABA) spectra ZrPc, the of phthalocyanineESI-MS analysis complexesgive a molecular (Figure ion S2, peak Supplementary at m/z~908. In Materials), the case of peaksbis (DABA) with theZrPc molecular bonded to mass one moremolecule than of exact DMF mass (bis of (DABA) complex ZrPc+DMF), could be seen peak due at to m one/z~981 or twois observed. molecules Signal of solvent at m (DMF)/z~950 extracoordinatedcorresponds to the to metalcomplex atom with of phthalocyanine one molecule complexof the solvent as presented (DMF) forwhile bis (PABA)two amino ZrPc groups in Figure are3. Coordinated solvent molecules are bonded more strongly with metal atom than axial ligand. Moreover, detached from the structure (bis (DABA) ZrPc+DMF-2NH2). The signal at m/z~880 relates to the loss asof ittwo is shown amino in groups Figure 3in, thebis structure(DABA) breaksZrPc complex. in ionization In ESI-MS, chamber there of the is spectrometer.a possibility of Fragmentation formation of startsradical with cations; removal especially of amine easily functionalities oxidizable from compounds, the ligand for followed example, by detachmenthydrocarbon of polyenes, phenone groupspolycyclic from aromatic the ZrPc hydrocarbons, structure. In thisporphyrins step, two and molecules so forth. of The DMF ions are observed bonded toby themass structure spectroscopy which willare quasimolecular be removed later ions during created ionization by the addition process. Inof thea hydrogen case of bis cation (DABA) [M+H] ZrPc,+ or the another ESI-MS cation, analysis for giveexample, a molecular Na denoted ion peak as [M+Na] at m/z~908.+ [45]. In the case of bis (DABA) ZrPc bonded to one molecule of DMF (bis (DABA) ZrPc+DMF), peak at m/z~981 is observed. Signal at m/z~950 corresponds to the complex with one molecule of the solvent (DMF) while two amino groups are detached from the structure (bis (DABA) ZrPc+DMF-2NH2). The signal at m/z~880 relates to the loss of two amino groups in bis (DABA) ZrPc complex. In ESI-MS, there is a possibility of formation of radical cations; especially easily oxidizable compounds, for example, hydrocarbon polyenes, polycyclic aromatic hydrocarbons, porphyrins and so forth. The ions observed by mass spectroscopy are quasimolecular ions created by the addition of a hydrogen cation [M+H]+ or another cation, for example, Na denoted as [M+Na]+ [45]. C 2020, 6, x 6 of 13 C 2020, 6, 1 6 of 13 C 2020, 6, x 6 of 13 H2N NH2

H2N O O NH2 O O

H C H C CH 3 O O CH3 3 O O 3 O O -32+2H O O N N N N H C H C 3 CH3 3 CH3 O O H C OO O H3C O Zr O CH -32+2H 3 Zr O CH3 N N 3 N N H C O H C O O 3 Zr N O CH3 3 Zr N CH3 N N N N N N N N N N N N N N N N N N N N N N N N N N N N m/z 1022N -210 Nm/z 992

m/z 1022 -210 m/z 992 H C HO OH 3 CH3 HO OH HNC HO OH 3 N CH3 HO OH -146 Zr H C N O 3 Zr O NCH3 -146 Zr H C O N 3 Zr O CH3 N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N N m/z 636 -123 m/z 782 m/z 636 m/z 782 -123

N N HN N N N NH N N HN N N NNH N N m/z~514 m/z~514 Figure 3. Fragmentation pattern of phthalocyanine complexes according to ESI-MS analysis. FigureFigure 3. Fragmentation3. Fragmentation pattern pattern of of phthalocyanine phthalocyanine complexescomplexes according to to ESI-MS ESI-MS analysis. analysis.

3.2.3.2. Spectroscopic Spectroscopic Spectroscopic Studies Studies and and Singlet Singlet Oxygen Oxygen Generation Generation OpticalOptical properties properties of ofPc Pc complexes complexes and and compositescomposites were were studied inin two two media media (DMSO (DMSO and and saline—solutionsaline—solution ofof sodiumofsodium sodium chloridechloride chloride in in in water). water). water). DMSO DM DMSOSO and andand saline salinesaline were were chosen chosenchosen for for for their their their compatibility compatibility compatibility in inbiological biologicalin biological systems. systems. systems. In thetheIn the UV-Vis-NIRUV-Vis-NIR UV-Vis-NIR absorption absorption absorption spectra spectra spectra of of MPcs, of MPcs, MPcs, two twotwo speciﬁc specificspecific regions regions are expected.areare expected.expected. Soret Soret Soret (or B)(or (or band B) B) bandusuallyband usually isusually seen is in isseen the seen rangein inthe the betweenrange range between between 300–400 300–40 nm.300–40 Band00 nm.nm. in Band theBand region in the between regionregion between 620–750between 620–750 nm620–750 named nm nm Q namedbandnamed is Q another band Q band is characteristic anotheris another characteristic characteristic feature. Both feature. feature. are seen Both Both in are theare seenspectraseen inin the of the spectraspectra studied of of the the complexes. studied studied complexes. complexes. The intense TheabsorptionThe intense intense is absorption visible absorption at 345 is nmisvisible visible (B band) at at 345 345 and nm nm 685 (B (B nm ba ba (Qnd)nd) band) andand 685 for685 bothnm (Q Pcs band)band) in DMSO forfor both (Figureboth Pcs Pcs4 a).in in DMSO Satellite DMSO peak(Figure(Figure is seen4a). 4a). Satellite at Satellite 618 nm. peak peak Similar is isseen seen positions at at 618 618 nm. nm. of theSimi Simi bandslarlar positions positions are seen ofof for the Pc bandsbands complexes areare seen seen in for saline—Bfor Pc Pc complexes complexes band at in340 saline—Bin nm, saline—B Q band band band at at 695 at340 340 nm nm, nm, and Q Q band satellite band at at 695 peak695 nm nm at and and 640 satellite satellite nm (Figure peakpeak4 b).at 640640 In nm transitionnm (Figure (Figure metal4b). 4b). In Incomplexes, transition transition metal complexes, these bands result from transition of electrons in molecular orbitals (d-d metalthese bandscomplexes, result fromthese transition bands ofresult electrons from intransi moleculartion of orbitals electrons (d-d transitions).in molecular B bandorbitals arise (d-d due transitions). B band arise due to deeper π-π* transitions and Q band is attributed to the π-π* transitions).to deeper π- πB* transitionsband arise and due Q to band deeper is attributed π-π* transitions to the π-π *and transition Q band from is attributed HOMO to LUMOto the ofπ-π Pc* transition from HOMO to LUMO of Pc rings. Change of shape of the Q band and shift of band transitionrings. Change from of HOMO shape of to the LUMO Q band of andPc rings. shift of Change band maxima of shape (main of the and Q satellite) band and correspond shift of band with maxima (main and satellite) correspond with face-to-face dimerization of phthalocyanine complexes maximaface-to-face (main dimerization and satellite) of phthalocyanine correspond with complexes face-to-face in water dimerization solution of and phthalocyanine conjugation of complexesπ-electron in water solution and conjugation of π-electron systems of two macrocycles. insystems water ofsolution two macrocycles. and conjugation of π-electron systems of two macrocycles.

(a) (b)

Figure 4. Absorption spectra(a) of studied phthalocyanine complexes in (a) dimethyl( sulfoxideb) (DMSO) and (b) saline. Molar concentration = 2 × 10−5 M. FigureFigure 4. Absorption 4. Absorption spectra spectra of studied of studied phthalocyanine phthalocyanine complexes complexes in (a)in dimethyl (a) dimethyl sulfoxide sulfoxide (DMSO) (DMSO) and (b) saline.and (Molarb) saline. concentration Molar concentration = 2 × 10−5 M.= 2 10 5 M. × − C 2020, 6, x 1 77 of of 13 C 2020, 6, x 7 of 13

The absorption spectra were also registered for different concentrations of complexes in DMSO The absorption spectra were also registered for diﬀerent concentrations of complexes in DMSO or or saline. The concentration of ZrPcs in DMSO was chosen to not exceed the value of Q band saline. The concentration of ZrPcs in DMSO was chosen to not exceed the value of Q band absorbance absorbance above 2.2 and the maximum concentration in saline used during experiment was about above 2.2 and the maximum concentration in saline used during experiment was about three times three times higher than in DMSO. The obtained spectra with the graphs showing the dependence of higher than in DMSO. The obtained spectra with the graphs showing the dependence of absorbance absorbance (at selected wavelength) on ZrPc concentrations are presented in Figure 5; Figure 6. (at selected wavelength) on ZrPc concentrations are presented in Figure5; Figure6.

(a) (b) Figure 5. Absorption spectra of different concentrations of bis(PABA)ZrPc in (a) DMSO (from 0.25 to Figure 5. 5. AbsorptionAbsorption spectra spectra of of different diﬀerent concentrations concentrations of ofbis(PABA)ZrPc bis(PABA)ZrPc in in(a) ( aDMSO) DMSO (from (from 0.25 0.25 to 2.2 µM) and (b) saline (from 0.25 to 6.0 µM) and the results of Beer-Lambert linearity experiment 2.2to 2.2µM)µM) and and (b) ( bsaline) saline (from (from 0.25 0.25 to to 6.0 6.0 µM)µM) and and the the result resultss of of Beer-Lambert linearity experiment (insets). (insets).

(a) (b)

Figure 6. 6. AbsorptionAbsorption spectra spectra of of different diﬀerent concentrations concentrations of of bis(DABA)ZrPc bis(DABA)ZrPc in in(a) ( aDMSO) DMSO (from (from 0.25 0.25 to µ µ 2.2to 2.2µM)M) and and (b) ( bsaline) saline (from (from 0.25 0.25 to to 6.0 6.0 µM)M) and and the the result resultss of of Beer-Lambert linearity experiment (insets). Capability of the complexes and composites as photosensitizers for singlet oxygen generation Capability of the complexes and composites as photosensitizers for singlet oxygen generation under irradiation was examined with 1,3-diphenylisobenzofuran which is a well-known reagent for this under irradiation was examined with 1,3-diphenylisobenzofuran which is a well-known reagent for purpose. Absorption spectra of Pc complexes and composites in DMSO were recorded before and after this purpose. Absorption spectra of Pc complexes and composites in DMSO were recorded before andaddition after ofaddition DPBF (Figuresof DPBF7 (Figures and8). Decrease 7 and 8). ofDecrease the DPBF of the absorbance DPBF absorbance (bleaching) (bleaching) ( λmax = 418(λmax nm) = 418 in and after addition of DPBF (Figures 7 and 8). Decrease of the DPBF absorbance (bleaching) (λmax = 418 the presence of ZrPc complexes and composites was registered under diﬀerent time of lamp irradiation. nm) in the presence of ZrPc complexes and composites was registered under different time of lamp irradiation. C 2020, 6, 1 8 of 13 CC 20202020,, 66,, xx 88 ofof 1313

(a) (b)

FigureFigure 7. 7. AbsorptionAbsorptionAbsorption spectraspectra spectra ofof of ((a (aa)) ) bisbis bis (PABA)(PABA) (PABA) ZrPcZrPc andand ( (bb)) bis bis (DABA) (DABA) ZrPc ZrPc solutions solutions in in DMSO DMSO beforebefore andand afterafterafter addition additionaddition of ofof 1,3-diphenylisobenzofuran 1,3-diphenylisoben1,3-diphenylisobenzofuranzofuran showing showingshowing DPBF DPBFDPBF bleaching bleachingbleaching when whenwhen exposed exposedexposed to light toto lightlightirradiation irradiationirradiation (due to (due(due singlet toto oxygensingletsinglet generation).oxygenoxygen generation).generation). (c) Irradiation ((cc)) IrradiationIrradiation time dependence timetime dependence ofdependence DPBF absorbance ofof DPBFDPBF in absorbanceabsorbancethe presence inin of thethe ZrPcs. presencepresence ofof ZrPcs.ZrPcs.

((aa)) ((bb))

Figure 8. Cont. C 2020, 6, x 9 of 13 C 2020, 6, 1 9 of 13 C 2020, 6, x 9 of 13

. (c)

Figure 8. Absorption spectra of (a) bis (PABA) ZrPc/GO(c) and (b) bis (DABA) ZrPc/GO suspensions before and after addition of 1,3-diphenylisobenzofuran showing DPBF bleaching when exposed to lightFigure irradiation. 8. Absorption (c) Irradiation spectraFigure of time (a 8.) bis Absorptiondependence (PABA) ZrPc spectra of /DPBFGO of and absorbance(a) (bbis) bis(PABA) (DABA) in theZrPc/GO ZrPcpresence/GO and suspensionsof ( bZrPc/GO) bis (DABA) ZrPc/GO suspensions composites.before and after addition of 1,3-diphenylisobenzofuranbefore and after addition of showing 1,3-diphenylisoben DPBF bleachingzofuran when showing exposed DPBF to light bleaching when exposed to irradiation. (c) Irradiation timelight dependence irradiation. of ( DPBFc) Irradiation absorbance time in thedependence presence ofof ZrPcDPBF/GO absorbance composites. in the presence of ZrPc/GO Photoluminescence excitationcomposites. and emission spectra of bis (PABA) ZrPc and bis (DABA) ZrPc in Photoluminescence excitation and emission spectra of bis (PABA) ZrPc and bis (DABA) ZrPc in DMSO are shown in Figure 9. The photoluminescence excitation spectra were recorded monitoring DMSO are shown in Figure9. The photoluminescence excitation spectra were recorded monitoring the maximum of ZrPc emissionPhotoluminescence band at 704 nm. Like excitation in the andcase emission of absorption spectr spectrum,a of bis (PABA) two broad ZrPc and bis (DABA) ZrPc in the maximum of ZrPc emission band at 704 nm. Like in the case of absorption spectrum, two broad bands were observed: oneDMSO in the are range shown of 300–400 in Figure nm 9. and The the photoluminescence second one above excitation 550 nm (here, spectra only were recorded monitoring bands were observed: one in the range of 300–400 nm and the second one above 550 nm (here, only the the satellite band around the620 maximumnm was recorded). of ZrPc emission To avoid bandan overlap at 704 of nm. emissi Likeon in band the case and ofexcitation absorption spectrum, two broad satellite band around 620 nm was recorded). To avoid an overlap of emission band and excitation wavelength, the 620 nm bandsexcitation were was observed: used (ins onetead in the of range690 nm of 300–400where the nm maximum and the second of Q bandone above 550 nm (here, only wavelength, the 620 nm excitation was used (instead of 690 nm where the maximum of Q band occurs). occurs). In the luminescencethe satellite spectra, band characteristic around 620 broad nm was band recorded). in the Torange avoid of an670–800 overlap nm of emissiwas on band and excitation In the luminescence spectra, characteristic broad band in the range of 670–800 nm was observed. observed. The measurementwavelength, conditions the did 620 not nm allow excitation to register was spectraused (ins oftead complexes of 690 innm saline where nor the maximum of Q band The measurement conditions did not allow to register spectra of complexes in saline nor composites. composites. occurs). In the luminescence spectra, characteristic broad band in the range of 670–800 nm was observed. The measurement conditions did not allow to register spectra of complexes in saline nor composites.

(a) (b)

FigureFigure 9. 9. ((aa)) Photoluminescence Photoluminescence excitation (λemem == 704704( anm) nm)) and and ( b)) photoluminescence spectraspectra ((λλexcexc =( b) 620620 nm) nm) of of bis bis (PABA) (PABA) ZrPc and bis (DABA) ZrPc in DMSO. Figure 9. (a) Photoluminescence excitation (λem = 704 nm) and (b) photoluminescence spectra (λexc = 4.4. Discussion Discussion 620 nm) of bis (PABA) ZrPc and bis (DABA) ZrPc in DMSO. Dimerization, higher order association and self-aggregation of Pc rings usually take place in Dimerization, higher4. orderDiscussion association and self-aggregation of Pc rings usually take place in polarpolar environments environments (polar (polar solvents) solvents) that that affects affects ph photochemicalotochemical and and photo photo physical physical properties properties of of Pcs, Pcs, forfor example,example, absorption absorption intensity intensityDimerization, of complexes of comple higher decreasexes order decrease during association dimerization during and self-adimerization of metalggregation phthalocyanine of of metalPc rings usually take place in phthalocyaninecomplexes. UV-Vis-NIR complexes. spectroscopypolar UV-Vis-NIR environments is a spectroscopy suitable (polar mean solvents) is to a study suitable that thisaffects mean effect ph [otochemicalto46 ].study Shifts this in and Qeffect band photo [46]. and physical properties of Pcs, Shiftschanges in inQ shapeband and of this changes bandfor indicate inexample, shape aggregation of absorptionthis band processes. indicate intensity Inaggregation order of tocomple utilize processes.xes Pcs indecrease biomedicine,In order toduring utilize metal dimerization of metal phthalocyanine complexes. UV-Vis-NIR spectroscopy is a suitable mean to study this effect [46]. Shifts in Q band and changes in shape of this band indicate aggregation processes. In order to utilize C 2020, 6, 1 10 of 13 phthalocyanines should have good solubility without aggregation in polar solvents [46]. Therefore, aggregation properties of the obtained complexes were studied at different concentration range (0.25–2.25 µM) in DMSO and (0.25–6.25 µM) in saline. First of all, the registered absorption spectra of the complexes are common for metal phthalocyanines in non-polar (DMSO) and polar (saline) solvents showing different width and relative intensities of the bands. Shape of the spectra shown in Figures5b and6b resulted from dimerization of Pc structures in aqueous solution. Looking at the spectra registered for various ZrPc concentrations, the findings were in agreement with Beer-Lambert law linearity experiment in DMSO showing that there is no clear evidence of molecules aggregation (dimerization and further agglomeration) in this solvent for the studied concentration range (insets in Figures5a and6a). The same should be for other organic solvents as verified in our earlier works [20,21]. In case of using saline solution for both complexes, deviation from Beer-Lambert linearity law was observed for ZrPc concentration higher than 4 and 3 µM, for bis (PABA) ZrPc and bis (DABA) ZrPc, respectively (insets in Figures5b and6b). Non-linear dependence indicated clearly high degree of molecules aggregation. Lower concentration limit for bis (DABA) ZrPc may be observed due to higher polarity of DABA ligands (higher tendency to dimerization than in the case of PABA) [20]. Eventually, in the spectra of GO-based structures functionalized with ZrPc derivatives, weak bands at around 690 nm were seen (Figure8a,b) confirming the presence of phthalocyanines in the composites. Photoluminescence excitation spectra (Figure9) showed that two ranges of light wavelengths can be used for samples excitations, that is, UV/blue and red/NIR. Red (here 620 nm) excitation confirmed the presence of red/NIR luminescence of ZrPc complexes. Photoluminescence was not recorded for complexes in saline or for composites. In the first case, the aggregation of ZrPc might be the reason for low luminescence properties. In the case of composites, low content of ZrPc on GO flakes (as indicated by the absorption spectra) would explain lack of seen light emission. Broadband red/NIR light emitting lamp was used for singlet oxygen generation tests. This commercially available light source was suitable for ZrPcs photoactivation. Both phthalocyanine complexes produced reactive oxygen species with similar effectiveness (Figure7). The DPBF absorption band was not seen after 2 min of irradiation. Despite the fact that the concentration of Pcs was much lower in the composites than in the solutions, singlet oxygen generation was still observed for these samples but the amount of reactive species was not enough high to react with the DPBF molecules after 4 min of irradiation. Nevertheless, the idea to link chosen Pcs to a GO carrier and to maintain their photoactivities was proved to be correct. The results shown in Figure8c marked that the activity of bis (DABA) ZrPc /GO is slightly 1 higher (faster DPBF bleaching). Differences in the amount of O2 generated by bis (L) ZrPcs may depend on their structure. Bis (PABA) ZrPc with two amino groups of two ligands is geometrically favorable (less hindrance for para position) but bis (DABA) ZrPc, with four amino groups in meta position, provides better connection/linkage to graphite oxide as support (mainly due to the possibility to form more hydrogen bonding but also through π-π stacking or covalent bonding to carrier surface). To increase the photoactivity of composites, one could think of loading more of Pc complexes on the GO structure. In this study, synthesis of composites was already performed in a way to ensure relatively high concentration of ZrPc and it would be difficult to obtain higher phthalocyanine content. But it must be noted that structure of complexes with different functionalities absolutely affects the 1 attachment process that further influence on the O2 generation as reported in our previous studies of other ligands bonded to ZrPc [38,47]. In conclusion, the generation of singlet oxygen has been a subject of great interest in different disciplines, from polymer science to medicine. High chemical reactivity of singlet oxygen can be utilized in biological systems, for example, in PDT [48] including antiseptic effect. Photoactivity capacity of the studied materials (both complexes and composites) was proved by the test of singlet 1 oxygen generation using DPBF. Comparison between complexes and composites showed better O2 generation in ZrPcs. From excitation and emission spectra of Pc complexes, one could conclude that C 2020, 6, 1 11 of 13 these phthalocyanine compounds are good candidates for PDT photosensitizers, since excitation at 600–700 nm and emission between 700–800 nm are essential to penetrate deep enough into tissues [38].

Supplementary Materials: The following are available online at http://www.mdpi.com/2311-5629/6/1/1/s1, Figure S1: TEM and SEM images of graphite oxide flakes with zirconium(IV) phthalocyanine derivatives; Figure S2: Mass spectra of (a) bis (PABA) ZrPc, (b) bis (DAPA) ZrPc; Table S1: IR wavenumbers and relative intensities of observed bands together with proposed assignment for the studied complexes and composites. Author Contributions: Conceptualization, Y.G. and A.L., methodology, L.T., Y.G. and I.T., investigation, L.T., Y.G., A.W., M.P. and A.L.; writing—original draft preparation, L.T.; writing—review and editing, L.T., Y.G., M.P. and A.L.; visualization, L.T., Y.G. and A.L. supervision, Y.G. and A.L.; project administration, A.L.; funding acquisition, Y.G and A.L. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by National Science Centre research grant No. 2016/23/B/ST5/024830. Conflicts of Interest: The authors declare no conflict of interest.

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