Photochemistry and Photobiology, 2021, 97: 930-935 Rapid Communication WD Check for updates Spectroscopic Analysis of Apoferritin Associates with the Water-Soluble Porphyrins ki Sakae*@® Department of Bioscience and Biotechnology, Fukui Prefectural University, Eiheij, Japan Received 29 May 2021, accepted 21 June 2021, DOI: 10.1111/php.13476 ABSTRACT TThe association behavior of apoferritin species with the anionic porphyrins, §,10,15,20-tetrakis(4-sulfonatopheny)porphyrin (LTPPS*) and its zincdID) complex (Zn TPPS™), which are ‘water-soluble even in acidic solutions, was investigated for the first time through UV-Vis absorption and fluorescence spec- troscopy in order to evaluate a potential ability of apoferritn as, a stimuli-responsive molecular capsule. The absorption maxi ‘mum wavelengths of both porphyrins were redshifted and the fuorescence intensity decreased, indicating the effective associ ation between apoferritin species and the porphyrins, although depended on the pHl adjustment procedure. At pH 2, the ZaiTPPS* associated with apoferritin subunits without demet- allating and protonating, while the free base porphyrin formed, the J-aggregate of the diprotonated species (HyTPPS”)q, with, extremely low fluorescence. As the concentration of apoferritin, Subunits increased, the HTPPS* subunits. associates, were formed accompanied by recovering the fuoreseenee. The asso- lation stoichiometries of 1 or 2 subunits/porphyrin obtained under neutral and acidie condi association constant of ZaTPPS' Stic interaction between apoferritin subunits and the por- phyrin under acidic conditions. INTRODUCTION Ferritin is a ubiquitous storage protein distributed in almost all, lives, Ferritin has 24 subunits of evo types, L:(light) and H (heavy) chains. These subunits constitute a nanocage structure \hieh can store up 10 4500 iron atoms inside, detoxify iron and, control the distribution in body (1). Iron-fiee ferrin is called apofertin. Apoterritin is not only used as a template for symthe- ‘is of metal nanoparticles, but also studied to develop functional healthy foods and novel cancer therapeutic agents (2-4). Thus, the investigation of molec sation into the apoteriin cage has been vigorously conducted in various fields of science. In order 10 encapsulate compounds into apoferiin, the main method isto utilize the property that apotemitin is reversibly di assembled and reassembled into subunits by pH conditions, i, apofertitin is disassembled into subunits under acidic conditions =Car aoa Nace 920 at fst, and afer adding a target compound, pit is neutralized to reconsruct the apolesitin cage and incorporate the target com Pound inside, The stimulisensitive openabefcloseable propety of the apofenitin cage is particulary alractve in drug delivery system (DDS), Furthermore, apoeritin has much atention a a nanocarir, since dhe hanced permeability and retention (EPR) effet ina cancer weatment is expected de to the outer diameter of about 12 ni (6). Porphyfin, which isthe bse structure of heme and chlor plust, exhibits spectoscopic properties depending on a solvation State of a porphyrin ring and aggregation. Moreover, porphyrins have been used as the detection reagent for trace metal ions dc to the central pocket of & porphyrin ring, where various metal ions can he accommodated. The solubility of porphyrin in water can be adjusted by the functional group at the meso position Owing to these properties, the effects of contal metals and func sional groups of porphyrins om the infleration between po phyrins and rani compounds can be investigated without changing their basic stractre and sic, the lat decades, porphyrins and related compounds have been used as photosensitizer in the photodynamic therapy such as HIV and cancer (6.7). At tat slag, since porphyrins are admin- istred as a relatively high concentration solution, aggregation could change the function of porphyrins and cause sie el Prhermore, the distribution of the thempeutc agents i vivo could be alecied by interacting with polymers. Therefor, the interactions of porphyrins with macromolecules such as proteins, dendrimers and PEG have boca studied for safe and effsetive formulations (8-10). Among porphyrins, zine) complexes have a Tong history as photosensitizer. Recently, the strctare and photoeacivity of the asociate of fertn wih the zinct) com pcx of protoporphyrin IX, which is a naturally occuring por Diyrin, have been studied (11). However, the carboxy group in the porphyrin sirictare is proton under aide conditions and the porphyrin bscomes insoluble in water (12). This poses the problem when the interaction betwoen apaferriin and porphyrin is investigated for understanding the encapsulation property of spoleritin as a pH-induced openieloxe controllable nanacarie Inthe present study, 510415. sulfonatophenyDporphyrin (HTBPS*), which Soluble even under acc conditions, was used. The interact beween the apoterrin cage and sebunits) andthe porphyrins could be investigated by means of UV-Vis absorption and fle rescence spoctwacopy. Nonmully, the inet) complex ZoTPPS'-) is demetallated and’ protonated under acidicconiltions; however, it was suppressed in the presence of apofer- fit species. In contrast to ZnTPPS*, HTPPS* was protonated and formed the Jaggregates. of the diprotonated species, (H.TPPS?>),, depending on the apoferiin subunits concentration. MATERIALS AND METHODS ‘A howe spleen apofersiin saline slution (0.138 me (= mol ay) NaCl (02 4m fered) was purchased from Sigma AUch (product number ‘A3GL1) and ased as received. The concentration of poten was ‘culated by using the molecular weight of the peptides (40 KDu). ‘The anionic porphyrins, 5.101520 ais slfoatophenyl pophyein UTPPS") disuonec acd tetakydrate (Dojindo) and the tetrsedium Salt of its zine(l) complex (NaZATPPS) (Frontier Seicatii), were ‘wed as recived. All samples were prepared 3s an aqueous solution. tn the investigation of the astoclaion Behavi bebween apo subunits and the porpyrin, apoferitin was decomposed ino subunits a pH 2 with HCL und then the porphyrin was added. In onder 10 Investigate a capability of eneapslaton into the cage, apfertin was composed int) subunits at pH 2 with HCL at fist then the porphyein ‘was sided, and finaly equimolar LOH was added to nevtralize and Sim LH,PO/LOH. butler solution was added 10 reconstruct te ‘poteriin cage at pll 7. Especially for ZaTPPS™, the samples were ucflly prepared to complete the reactions and confined steady ‘se, since the dometalltion of the zine) center oseurs within 30 ain (8, UV-Vis amorpion and fluorescence spectra of the aqueous sample Solutions were messured with an UV-Vis spectrophorometer (ASCO, V.630) anda orescence spectrphotometer (itachi, F-7000), respostvely. The absorption spectra were comected y subrating he abwerbance of the conesponding apofentin conceaations. The Thuorescence spc ere taken st an excitation maximum in cach system RESULTS AND DISCUSSION pH elfect on the association between apoferritin and porphyrin Apoferrtin nanocage structure reversibly disassembles into 24 subunits at pH < 3.40 (13). The pH-triggered structural change has the potential as a stimulisesponsive molecular capsule. The intermolecular interaction between apoteritin (or subunits) and the anionic water-soluble porphyrins in the aqueous solution was cd through the UV-Vis. absorption spectroseopy. shows the typical UV-Vis absorption spectra of the free presence of equimolar apoferrtin under acidic and neural pH conditions Both porphyrins are demetllized and protonated 1o generate the diprotonated. species, HJTPPS?-, whose the Soret and Q hands locate a 434 and 648 nm at pH < AS in the absence of apoterstin (9). HeTPPS* was regenerated when pH was ‘ujusted to the neutral and the Soret and Q hands are observed ft 413, 515, 882, S80 and 632 am, The absorption maximum ‘wavelength at the Soret band of HETPPS* (413 nm) was signifi cantly redsifted to 417 nm in the presence of apolestin at pH 72 (Fig. 1a, black line). In the ease of ZaTPPS" the shoulder response appeared around 428 nm with the redshift from the inns Soret and at 421-431 nm (Fig. a, blue line). Is also ‘work noting that the Q hands of both porphyrins. were not degenerated but slightly redshifed from that of the exginal por phyrins. These spectral changes demonstrated the effective asso ciation between apoferrin and the porphyrins in the aqucous solution. On the other hand, the spectra of the porphyrins added under maintaining. neutral conditions without disassembling the Photochemistry and Photobiology, 2021, 97 931 (a) 06-5 T T T 0.4 Abs 400 500 600 wavelength /nm 700 () 0.6 > +++ Abs 500 600 wavelength /nm © 06-4 1 T r 500 600 wavelength / nm Figure 1. lt dependences of absorption spect of the fse Base por plyrin (Black lines) and the zine) complex (hue lines) inthe absence {ore ins) and presence (solid lines) of apotetin spies. (a) pH 72 ‘with disassembling and reassembling apofewin cag, (b) pH 72 main taining the apofentin eage and (c) pH 21. The concenation of apace sin athe poophytins was Ty. 700 poterrtin cage into subunits under acidic conditions were handly ‘modified from those of the porphyrin alone (Fig. 1b). These spectroscopic results Jemonstated that the porphyrins coukd not992 Hiroki Sakae associate withthe peripheral region of the apofertin cage and pofettin need to be once disassembled into subunits in onder to interact withthe porphyrins. As described abose, bath por phyrins hocome H.TPPS*~ under acide eanltions; however the coexistence of apoferitin subunits provided the spectra indicat- ing the Soret band at 421 nm for the fee base porphyrin and 429 nm for the zine) complex (Fig. 16). The spectral resus suggested thatthe associating species ofthe porphysins with apor fern subunits were not HTPPS™, but mainly HLTFPS" and ZoTPPS'™. Nevertheless, it should be note that the Soret (484 nm) and Q (702 nm) bands of the Jaggrezaes of diprotor rated spovies, (HJTPPS" slighty appsared in the ree base Porphyrin system. The "similar aggregation behavior of HyTPPS~ monomers inthe presence of the dendrimer and PEG has boon reported (8,14). This behavior at pH 2.1 and the inton- sity ofthe shoulder response at pH 7.2 depended on the concen- tation of apoferriin species are described in the following section. Apoferritin concentration dependence of the association behavior Figure 2 shows the apofertitin concentration dependence of the 1 pM porphyrins absorption specra at pH 7, “The intensities of the inttnsie Soret band of both poxphytins were decreased with increasing the concentration of apoferitin, followed by the redshift of the absorption maximum wavelength, ‘The Q bands indicated similar shifis. The comesponding decrease was also observed in the fluorescence intensity (see Figure SI). The well-defined isosbestic point in the Soret band region was porphyrin is quantitative. In order to determine the association ‘constant (A), the following reaction was assumed, P-4nS2P5,, K =|PS,/[PIS!" o where P, $ and PS are the free (nomassociating) porphyrins, apo- ferritin subunits and the porphyrin-subunits assocfates, respec tively, and m is the number of apofeeriin subunits associating with the porphyrin, It should be noted that $ is apoferitin su units, since apoferitin is disassembled into subunits duri interaction with the porphyrins. When the molar fractions of P tnd PS, are described as ap and ay, for instance, the absorbance at 431 nm (Ags) for the ease of Zn TPPS* is expressed as Aust = pp + yen ° ‘This equation can be rewriten by. Aw ° where + is the concentration of spofertin subunits. In the ease OF the free base porphyrin, the isosbesic point was obtained at 4417 am, However, 28 described below, HyTPPS® and its J+ tes were fomned in the presence of the lower concent tion of apoferitin subunits under acidic conditions. The apoter- ttn cage is reassembled, and (H,TPPS™), and HyTPPS* are nerated to HeTPPS* when pH is readjusted to 7. Since the free hase porphyrin species complicatedly changed in that maa nor depending on pls and the apoteritin subunits concen tions, the K value was not detennined by applying Eq. (3) wo the ‘esperimental data. The best fit is shown ia Fig. 2b, and Table 1 sommarizs the obtsined fitting parameters. exhibited at 427 nm for ZnTPPS' system. The results cal io The revuls suggesed that 1 apofertin subunit associated the conclision that the association hetwcen apoferriin and the with 1 ZnTPPS* molecule under the neutral condition @ 06 ©) 08 3 oat 3 et jee en 500 600 700 ‘060 100 160 200 wavelength / nm [Apoferstin subunits) / uM © 06 —___. © os-—>—]+ oa | gostee 7 2° | 2 oak 7. o4 7 (ere repenen 400500600700 ‘050 100 150 200 wavelength / nm Figure 2 Apoferitia conconration dependences of absorption spect and the absorbance measured ia (a,b) ZATPPS'™ and (,d) H,TPPS* (Apoferritin subunits} / uM systems sc pH 7.0-7.2. The concention ofthe porphyrins was I yx. The concentration of apfentitin increased fom Ot 10 ys (eotesponding 49 240 pw apo Feritnsubunis)b) The solid line eter to the iting cuve obained fom Ea, (3)fable 1. Binding parameters for the intrction between apotersiin spe ces and the porphyrins obsined fof experimental data using Fa. () ZAP ee re! oe * Ke" 2 Lo2 17203 1S40t On LOM 7 10202 1200 . 2 Crichton etal have reported that the protoporphyrin IX dive tives bind to apoferrtin with a maximum stoichiometry of 0.5 for 1 porphyrinsubunit, respectively, that comespond 10 12 or 24 porphyrinvapofertin (15). Although the present results. were in good agreement with the previous study in terms of the Finding stoichiometry. they measured the absorbance by the porphyrin titration with the fixed apoferitin concentration under the certain pH conditions, whece apofertin ean maintain a cage structure, On the other hand, we have previously reported that the addition of the ferritin cage under the neutral condition handy affected the fluorescence spectrum of 8anilino-1- naphihalenesulfonate, which is often used a a fluorescent probe to investigate protein folding (16). In the present study, no Significant diferences of the absorption spectra were observed between the porphyrin alone system and the apoferritin coexis- tence system in the similar tiation method 10 Crichion’s one (Gata not shown) Tis i probably due to the Functional group and the meta iron center of porphyrin species, since apoferitin is involved a Storage and release of iran in vivo, where iron atoms bind to heme, which has a protoporphyrin IX. stucture ‘The siructural and mechanistic analysis and effeets of functional groups and central metals of porphyrins on the interaction between the apotertin eage and the porphyrins will be invest gated in more detail in the future Figure 3 shows the absorption speetra of the 1 pw porphyrins a diferent apoferitin subunits concentrations at pH 2, AAs described in above section, the diprotonated [1TPPS?~ is generated in the absence of apoferriin species. Since the Q band region reflects the structural characteristics of the por Dhytin species, the demetallaton, protonation and aggregation processes could be traced. In the zine(H}) porphyrin system, the Q bands at 595 and 645 nm tributed to H,TPPS™ monomer ‘were mainly observed below the S/P (= [Apoferritin subunits) [Porphyrin)) ratios of 0.1. At the higher SIP ratios, these Q Thands disappeared and thoxe of Za TPPS*~ (589 and 599 nm) ‘were reconstructed withthe shift ofthe Soret band from 434 to 429 nm. The absorbance at 429 nm became constant when the SIP ratio was 12 oF more. The corresponding fluoreseence spee- tral changes were also observed, ic. the fluorescence spectra of HTPPS® monomer with the maximum wavelength at 665 nm converted to those of ZsvTPPS* with the maximum wavelength a1 606 nm as inereasing the concentration of apofertin subunits 24). The spectral changes demonstrated that ZaTPPS* could he protected from the demetalltion and proto- nation in acidic solutions inthe presence of apofettin subunits (On the other hand, inthe ease of the fee base porphyrin, the cral changes were significantly dependent on the SIP ratio. the ease of the zine(ll) porphyrin, the Soret and Q Photochemistry and Photobiology, 2021, 97 933 bands mainly duc to H{TPPS'~ monomer were observed below the SIP ratios of 0.1 decreasing in the absorbance of these bands, while the slight absorption peaks appeared at 491 and ‘700 nim attributed 10 the J-aggregates of HTPPS?-. Although the intensity of these bands inereased and the J-aggregates for- ‘mation proceeded at the SIP ratios of 0.1 to 0.5 with the drastic seerease in the fluorescence intensity (soe Figure S2h), these Fhands began to deerease accompanied hy the inerement and blueshift of the Soret band from 434 to 420 nm when the SIP ratio. was higher than 0.5. In addition to these changes, the Q bands of the HyTPPS* were reconstructed and the Muoresceace intensity at 651 nm recovered ahove the SIP ratios of 2.4. The Soret and Q bands of H{TPPS*~ monomer and its Jagerevates, ‘were almost disappeared when the SIP ratio of 48. The depen- dence of absorption and fluorescence spectra of the free base porphyrin on, the apofemitin subunits concentration suggested that HyTPPS'™ monomer associated with apofertin subunits, ‘was formed as the concentration of apoferritin subunits, increased. The similar spectral transition has been reported in the spectroscopic study of the interaction between H3TPPS* and human serum albumin (10). In the fe base porphyrin sys- tem compared to the zinc(I) pophyrin one, even when the SIP ratio. was high, the fluorescence intensity was weakened with the redshift from tho intrinsic fluorescence spectrum of HTPPS*, indicating that HTPPS*. H,TPPS* monomer, and its J-aggregates cooxisted, Equation (3) was applied 1 the absorbance at 429 am for ZaTPPS* -subunits associates (0) rd at 420-nm for HTPPS'subunils associates. (Aya) exclude ing the low concentrations of apoferiin subunits (Fig. 3b), “The parameters obisined from the fing analysis of the exper ‘mental data are shown in Table 1. In audition to the aforemen- tioned investigation by Crichton et al. (15), it has also been, reported that one metal-containing protoporphyrin IX is sand= wiched among the dimer of apoferriin subunits (17). The fbiained value of m indicated that 1 or 2 apofersitin subunit(s) could associate with 1 molocule of the porphyrins. The K value for ZnTPPS* was quite higher than that of HSTPPS* under acidic conditions, suggesting the importance of the coordination, to the zine(D) center as well a the electrostatic interaction with, apoferrtin subunits. ‘The effects of sll and its concentration on the associate for- roslls, it was suggested thatthe interaction esween apoferiin subunits and the porphyrins was « key in the formation of asso- ciates. Therefore, various salts, ie. LICL, CaCl, KCL and NaCl, were added to acidic solutions, where apofeeritin disassembles, into subunits. In the zinc(D) porphyrin system, the addition of fany salts hardly affected the absorption spectra. On the other hhand, in the free base porphyrin system, the spectrum of the HTPPS*~ monomer was reconstructed from that of its gates at the high salt concentration, especially when using ‘These spectral changes were not duc tothe type of cation, but to the anion concentration. In other words, the large amount ‘of CI” canecled the positive charge of apoferitin subunits, weak- cning the electrostatic interaction between the subunits and the porphyrin. Furthermore, the results also demonstrated that the ine(l}) porphyrin formed the stuble associates. with apoferiin subunits compared to the fre hase porphyrin934 Hiroki Sakae @ Abs ©) 08-4 Absez9 400 500 600 700 wavelength / nm © asc T T ) ol 400 500-600 700 wavelength / nm fy borers ieee 0 10 2 30 40 [Apoferrtin subunits] / uM Figure 3. Apoertin subunits concentration dependences of sbsogpion spectra and the absorbance measured in (a,b) ZaTPPS* and (cd) HTPPS* systems at pH 20-2. The concentration of the porphyrins was T pu, The concentration of apoferitn sabi increased fom 0 9 48 ya (omespond ing 0 2 jo apofertn). (bd) The soi ine refer to the iting eve obs frm Ex. (3), CONCLUSION Molccular encapsulation properties of apofertin uiilizing pl triggered reversible disassemble and reassemble into. subunits were studied for the first time through UV-Vis absorption and joreseence spectroscopy by using the porphyrins, which are water-soluble even under acidie conditions. The interaction with apoferrtin subunits was @ key step in the encapsulation process fof the porphyrin molecules into the apoferitin cage. It was also demonstrated that the central zinc(II) metal of porphytin played fan important role in the stability of the apoferrtn species-por- phyrin associates. The apoferitin species acted as & protective ‘agent against the zinc() porphin, while promating the forma- tion of aggregates of the free base porphyrin, Furthermore, i ‘was revealed that the aggregation and emission properties of the porphyrins can he controlled by the concentration of apoferrti, ‘The porphyrins bound to the apoferrin species more strongly in ‘acidic than in neutral solutions, This suggests thatthe porphyrins ate likely (0 be released from apoferritin, for example, in blood, (pH = 7.4). Therefore, apoferritin can be used as a nanocarrier in a functional DDS by studying the encapsulation of ‘various drugs not limited to porphyrins into apotertin AcknowledgemonsThis research was financially supported by the Sratapie Resareh Promosion Gast (02R020214) from Fukui Prefectural University. CONFLICT OF INTEREST ‘The authors declare that they have no knowin competing financial interests of personal relaionships that coukl have appeared to influence the work reported in this paper. Data Availability Statement ‘The datasets generated during and/or analyze during the current, study are available from the eoresponding author on reasonable request SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section at dhe end of the acl Figure SI. Fluorescence specira of (a) ZnTPPS* and (0) HESTPPS*™ in the presence of apoferritin at pH 7.1-7.2 Figure $2. Fluorescence spectra of (a) ZnTPPS* and (6) HSTPPS* in the presence of apofersitin subunits at pH 2.02.1, Figure $3, UV-Vis absorption spectra of (a) ZaTPPS'~ and (b) HyTPPS* in the presence of apofetitin subunits and various chloride salts at pF 20-21 REFERENCES 1. Theil, E. C. (1987) Fein: strict, gene regulation, and cela function i animals, plans, and micoorgnisms. Annu Rev. 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