pubs.acs.org/cm Article Supercritical CO2 Synthesis of Porous Metalloporphyrin Frameworks: Application in Photodynamic Therapy Márta Kubovics, Oriol Careta, Oriol Vallcorba, Guillermo Romo-Islas, Laura Rodríguez, Jose A. Ayllón, Concepción Domingo,* Carme Nogués,* and Ana M. López-Periago* Cite This: Chem. Mater. 2023, 35, 1080−1093 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: A series of porous metalloporphyrin frameworks prepared from the 5,10,15,20-tetra(4-pyridyl)porphyrin (H2TPyP) linker and four metal complexes, M(hfac)2 M = Cu(II), Zn(II), Co(II), and Ni(II) (hfac: 1,1,1,5,5,5-hexafluoroacetylacetonate), were obtained using supercritical CO2 (scCO2) as a solvent. All the materials, named generically as [M-TPyP]n, formed porous metal− organic frameworks (MOFs), with surface areas of ∼450 m2 g−1. All MOFs were formed through the coordination of the metal to the exocyclic pyridine moieties in the porphyrin linker. For Cu(II), Zn(II), and Co(II), incomplete metal coordination of the inner pyrrole ring throughout the structure was observed, giving place to MOFs with substitutional defects and leading to a certain level of disorder and limited crystallinity. These samples, prepared using scCO2, were precipitated as nano- to micrometric powders. Separately, a layering technique from a mixture of organic solvents was used to crystallize high-quality crystals of the Co(II) based MOF, obtained with the formula [{Co(hfac)2}2H2TPyP]n. The crystal structure of this MOF was elucidated by single-crystal synchrotron X-ray diffraction. The Zn(II)-based MOF was selected as a potential photodynamic therapy drug in the SKBR-3 tumoral cell line showing outstanding performance. This MOF resulted to be nontoxic, but after 15 min of irradiation at 630 nm, using either 1 or 5 μM concentration of the product, almost 70% of tumor cells died after 72 h. 1. INTRODUCTION building blocks in MOFs involves the use of Hf(IV)13 and Porphyrins are key building block precursors in many areas of Zr(IV)14 as metal centers for head and neck cancer and synthetic chemistry. As an important example, the preparation cervical cancer, respectively. of metal−organic frameworks (MOFs) containing porphyrin Among the most studied porphyrin MOFs are those linkers is currently of great interest as they can form involving Zr(IV), giving high stability and structural diver- coordination networks with multiple applications such as sity,15 Ti(IV), applied in photocatalysis,16 and divalent Zn(II), catalysis, sensors for molecular recognition,1−3 or drugs for Co(II), and Cu(II) cations, forming usually very stable 2D cancer treatment.4−6 In the latter, porphyrins are the most sheet-like structures.17−19 Less common, but equally func- extensively used material for photodynamic therapy (PDT).7 tional, are the porphyrin-based MOFs with pyridyl motifs that The PDT procedure is based on the local application of a can form supramolecular structures through self-assembly.20 In photosensitizer in the affected area that, by light radiation of a particular, 5,10,15,20-tetra(4-pyridyl)porphyrin (H2TPyP), the certain wavelength, induces the formation of reactive oxygen linker chosen in this study, possesses several potential species (ROS)8 able to destroy the harmful cells through either coordination sites. The inner tetratopic unit can be coordinated by displacing the two protons of the N−H necrosis or apoptosis.9 In this technique, the use of net pyrrole subunits, and the exocyclic four pyridyl groups linked porphyrins is often limited by their low biostability in aqueous to the porphyrin ring can potentially coordinate from one to media, self-aggregation, and nonselective tumor targeting.10 Some of these drawbacks can be significantly attenuated by building porous structures in which the porphyrins are the Received: October 3, 2022 organic linkers located between metal-containing species, thus Revised: January 17, 2023 obtaining porphyrin-based MOFs. From them, the so-called Published: January 30, 2023 fourth generation of photosensitizers is built,11 where the porphyrin moieties are somehow isolated and their aggregation is hindered.12 The first report on PDT using porphyrins as © 2023 The Authors. Published by American Chemical Society https://doi.org/10.1021/acs.chemmater.2c03018 1080 Chem. Mater. 2023, 35, 1080−1093 Chemistry of Materials pubs.acs.org/cm Article four metal ions, which can greatly enrich the structure and Finally, the scCO2 precipitated Zn(II) MOF was successfully functionality. Nevertheless, not many examples of MOF tested as a potential photosensitizer in PDT. constructions are found in the literature for this type of pyridyl-based porphyrin linkers, presumably because H2TPyP 2. MATERIALS AND METHODS has an extremely low solubility in most conventional liquid 2.1. Materials. The organic linker 5,10,15,20-tetra(4-pyridyl)- solvents, which is difficult enormously for their processing. In porphyrin; the metallic units of copper, zinc, cobalt, and nickel fact, H2TPyP is only partially soluble in chloroform, hexafluoroacetylacetonate ([Cu(hfac)2]·xH2O, [Zn(hfac)2]·2H2O, dimethylformamide, and trichloroethane. Examples of total [Co(hfac)2]·xH2O, and [Ni(hfac)2]·xH2O); the solvents chloroform metal complexation are the coordination of Zn(II) ions with (CHCl3), methanol (MeOH), and 1,1,2-trichloroethane (Cl3Et); and the pyridyl groups of adjacent H2TPyP molecules and the same sodium anthracene-9,10-dipropionic acid (Na-ADPA) and perinaph- structure but with Ag(II) coordinated to the pyrrole units thenone, used for the singlet oxygen measurement, were all purchased forming 2D coordination polymers.21 Two other examples of from Sigma-Aldrich. CO2 (99.995%) was supplied by Carburos Metálicos S.A., Air Products Group (Spain). full coordination in TPyP2− were obtained using Cu(II) and 2.2. Methods. 2.2.1. Synthesis of MOFs. 2.2.1.1. scCO2 Syn- consisted of porous 2D coordination networks involving the thesis. [Zn-TPyP]n, [Cu-TPyP]n, and [Ni-TPyP]n MOFs were paddlewheel Cu(II) tetraacetate [Cu2(AcO)4] secondary obtained in pure scCO2. Reactions were performed in a 10 mL vial building unit22 or the Cu(hfac)2.23 In both cases, Cu(II) placed into a 100 mL high-pressure autoclave with two opposite fulfilled different tasks since it acts as a metal source to sapphire windows (Thar Design). The vial was charged with 0.34 mol metalize the porphyrin ring by interchanging Cu(II) with the of the metal complex M(hfac)2 and 0.11 mol of the organic linker, two central protons of the NH pyrrole ring and as a neutral standardizing a molar ratio of ca. 3.1:1 for M(II)/H2TPyP in all cases. A small magnetic bar was also added, and the vial was finally capped node maintaining the ligands AcO or hfac. with cellulose filter paper. In case of [Co-TPyP]n, ∼5 v % of CHCl3 The described H2TPyPs-based MOFs have been obtained was also added as a cosolvent. Experiments were performed using using long-lasting synthetic methods (from weeks to months), scCO2 at 20 MPa and 60 °C, stirring the powder into the vial at 500 either using layering (solvent diffusion) or solvothermal rpm, and for 72 h. After this running time, samples were cleaned with approaches. These techniques produce high-quality crystals a flow of fresh CO2 at high pressure to remove the excess of unreacted proper for single-crystal X-ray diffraction (XRD) resolution but M(hfac)2. Finally, the reactor was slowly depressurized and cooled in extremely small yields. Besides, these approaches can have down to room temperature, to recover in all cases a fine garnet- the limitation of solvent incorporation into the pores of the colored dry powder, in an average yield of 85 wt %. 2.2.1.2. Layering. Crystallization assays using the layering approach MOF, which can modify the structure of the final product. were tried for all studied metals. However, only the one involving Currently, the synthesis of MOFs is beyond the study of their Co(II) successfully produced crystals available for structural fundamental framework as in many circumstances, developing elucidation. For this experiment, H2TPyP (0.025 mmol) was key applications does not necessarily imply having well- dissolved in 10 mL of Cl3Et/MeOH (3:1) and layered with 2 mL resolved crystal structures. In fact, amorphous or semi- of fresh MeOH. A solution of [Co(hfac)2(H2O)x] (0.104 mmol) in amorphous MOFs have also shown a high capability to 10 mL of MeOH was carefully layered onto the pristine MeOH layer. perform certain applications, such as in drug delivery After several weeks at room temperature, purple needle-like crystals precipitated. The crystals were carefully filtered, rinsed with fresh systems.24 In this way, the search for new preparation methods MeOH, and finally dried under vacuum. involving short reaction times, low temperatures, and avoiding 2.3. Characterization. The structure of the obtained samples was toxic solvents, while achieving large yields, is under strong analyzed by powder XRD (PXRD) using a Siemens D5000 X-ray surveillance, even if the quality of the crystals is partially powder diffractometer using the Cu Kα incident radiation. The compromised. Among the different techniques developed to diffraction patterns were recorded from 2θ = 5 to 30°, with a step scan synthesize MOFs and coordination polymers, those based on of 0.02° counting for 1 s at each step. The morphological analysis was supercritical CO2 (scCO2) have proven to be excellent for performed using a Quanta FEI 200 FEG-ESEM scanning electron precipitating materials in high yields. scCO2 has been microscope. The thermal properties of the samples were evaluated by thermogravimetric analysis (TGA, PerkinElmer 7). Measurements traditionally used for removing entrapped solvents within were carried out under a N2 atmosphere, raising the temperature at a MOF structures. However, in the past few years, it has also rate of 10 °C min−1. Fourier transform infrared (FTIR) spectra of the been used in our research group as a solvent for the synthesis solid samples dispersed in KBr were recorded on a PerkinElmer of MOFs, either by using exclusively scCO2,25−27 or adding a Spectrum apparatus. The textural properties were determined by N2 cosolvent,28 with interesting applications in medicine (bio- adsorption/desorption experiments, performed at −196 °C, using MOFs),29 sensors30 or gas adsorption after hybridizing with ASAP 2020 Micromeritics Inc equipment. The samples were graphene aerogels.31 In essence, the use of the scCO2 previously degassed at 120 °C for 24 h. The Brunauer−Emmet− technology has proved excellent to prepare materials for Teller (BET) surface area (SBET) of the precipitated compounds was estimated in the relative pressure range of ca. 0.05−0.20. The biomedical applications.32 In the search for new applications, micropore Langmuir surface (SLangmuir) was also calculated, together and as a part of ongoing work, we have explored here the with the micropore volume (Vmp) estimated by the t-method. possibilities of precipitating MOFs based on the H2TPyP Elemental analysis was performed using a Flash EA2000 Thermo porphyrin linker in scCO2. Four metal hexafluoroacetylaceto- Fisher Scientific analyzer. Ultraviolet−visible (UV−vis) absorption nate complexes [M(hfac)2, M = Cu(II), Zn(II), Co(II), and measurements in water were carried out using a Varian Cary 5000 Ni(II)] were used to complete the MOF structure, using only apparatus with an operational range in the spectrophotometer of scCO2 as a solvent or adding a small amount of chloroform as 190−3300 nm. Dynamic light scattering (Zetasizer Nano ZS Malvern Inst.) was used to measure the hydrodynamic size. a cosolvent, when necessary. In order to obtain crystals for the Single-crystal XRD (SCXRD) experiments for [{Co- structural elucidation, a layering approach was attempted for all (hfac)2}2TPyP]n were performed in the XALOC beamline at the the studied products. However, this approach only allowed ALBA synchrotron (Spain). Data were collected at 100 K with a attaining proper crystals for the MOF involving Co(II), which 0.72931 Å wavelength using the Dectris Pilatus 6M detector placed at was further elucidated in regard to the crystalline structure. 120 mm from the sample. Nine scans were performed from 0 to 360° 1081 https://doi.org/10.1021/acs.chemmater.2c03018 Chem. Mater. 2023, 35, 1080−1093 Chemistry of Materials pubs.acs.org/cm Article in steps of 0.5° with a collection time of 0.15 step−1. The scan was were washed four times with HBSS to remove any product remains, repeated at three different N angles (0, 45, and 90°) and merged followed by fresh medium addition. Later, cells were either kept in afterward to increase the completeness and redundancy when dark conditions (not irradiated) or irradiated for 15 min using a possible. Data were indexed, integrated, and scaled using the XDS PhotoActivation Universal Light device (PAUL, GenIUL) in the software.33 The crystal structures were solved by intrinsic phasing and range of 620−630 nm (red light) and with a mean intensity of 55 mW refined with SHELXL (version 2014/7)34 using Olex2 as a graphical cm−2 (light dose of 33 J cm−2). Cell culture viability assessments were interface.35 performed twice, right after irradiation (viability at 24 h) or after 2.4. Measurement of Singlet Oxygen (1O2) Production. being incubated for an additional 48 h in standard conditions 2.4.1. Singlet Oxygen Detection via Fluorescence Decay of ADPA. (viability at 72 h), using the Alamar Blue assay. Three independent Singlet oxygen formation was detected using a Cary Eclipse experiments were performed for each condition. spectrofluorometer (Agilent). The process involved measuring the 2.5.3. Alamar Blue Assay. Cell viability was determined using the fluorescence decay of ADPA upon irradiation in the presence of the Alamar Blue cell viability reagent (Thermo Fisher Scientific). In brief, [Zn-TPyP]n photosensitizer.36 For this, 100 μL of a Na-ADPA water after product incubation either during 4 or 24 h, the cell medium was solution (1.2 mM) was added to 3 mL of [Zn-TPyP]n dispersion in removed, and cells were washed four times with HBSS before adding water (0.15 mg mL−1). The mixture was irradiated at 630 nm for 15 10% Alamar Blue to the fresh medium. Cells were then incubated for min using the same light source as for the photodynamic treatment 4 h in standard conditions in the dark. After incubation, the medium (PhotoActivation Universal Light PAUL device, GenIUL). After each was collected, and 200 μL of the solution was transferred to a black- 5 min, the mixture was introduced to a quartz cuvette of 1 cm, and the bottom Greiner CELLSTAR 96-well plate (Sigma-Aldrich). Finally, fluorescence emission spectra of the mixture were recorded in the the fluorescence of the medium was measured at a 590 nm 380−600 nm range, using an excitation wavelength of 355 nm. wavelength after excitation at 560 nm on a Spark multimode 2.4.2. Singlet Oxygen Quantum Yield (ϕΔ) Measurement. Room- microplate reader (Tecan). Three independent experiments were temperature singlet oxygen phosphorescence was detected at 1270 performed for each condition. nm with a Horiba-Jobin-Ybon SPEX Nanolog spectrofluorometer 2.5.4. Product Internalization. To visualize if there was any using a DSS-IGA020L detector. The use of a Schott RG 1000 filter product internalization, 1.75 × 105 cells were seeded on confocal 35 was essential to eliminate from the infrared signal all the first mm dishes provided with a glass coverslip bottom. After 24 h of cell harmonic contribution of sensitizer emission in the region below 850 seeding, 1 μM product was added to the culture. After product nm. The singlet oxygen formation quantum yield was then incubation (4 or 24 h) in standard conditions in the dark, cells were determined by direct measurement of the phosphorescence at 1270 washed four times with HBSS to remove the noninternalized product nm following irradiation of the aerated aqueous dispersions of the and incubated with 5 μL of wheat germ agglutinin (WGA) 448 for 15 samples. Perinaphthenone in dichloromethane was used as a standard min to detect the limit of the cells. Then, images were acquired using reference, applying eq 1. a Leica TCS-SP5 AOBS spectral confocal laser scanning microscope (Leica Microsystems) using a PlanApochromatic 63× objective lens. ref emission1270 nm = WGA-Alexa Fluor 488 (plasma membrane) and product excitation ref emission1270 nm (1) was carried out using 405 and 488 nm laser lines, respectively, using a sequential mode. Different PMT devices [600−763 nm for product ref where Δ is the singlet oxygen formation quantum yield of the fluorescence emission and 500−550 nm for WGA fluorescence reference compound ( ref = 0.79). emission (membrane)] were used to detect each corresponding 2.5. Cell Culture Experiments of [Zn-TPyP]n. Biological spectral range. A series of images were further analyzed with Fiji experiments were performed using the SKBR-3 cell line, a tumorigenic software (ImageJ−NIH). human mammary epithelial cell line (ATCC, USA). Cell culture was 2.5.5. Statistical Analyses. The statistical analysis was performed carried out in McCoy’s 5A modified medium (Gibco) supplemented using GraphPad Prism version 6.01 for Windows (GraphPad with 10% fetal bovine serum (Gibco) in a 37 °C humidified incubator Software). Quantitative results were analyzed using a two-way set to 5% CO2 (standard culture conditions). For both analysis and ANOVA with a minimal significance level set at p ≤ 0.05. In the toxicity evaluation in the dark and under irradiation (photodynamic figures, significance is represented by an asterisk, which means that experiments), cells were seeded in 4- or 24-well plates at a density of the values are significantly different from their control (p < 0.05). 3.5 × 104 cells per well. For confocal laser scanning microscopy (CLSM) experiments, cells were seeded in special confocal 35 mm 3. RESULTS AND DISCUSSION dishes provided with a glass coverslip bottom (μ-Dish 35 mm, high Glass Bottom, Ibidi GmbH) at a density of 1.75 × 105 cells per well. 3.1. scCO2 Synthesis. It is generally recognized that when All product treatments were performed 24 h after cell seeding. Stock preparing MOFs, the solvent of choice is highly important product dispersions were prepared in filtered distilled water to a final since it can affect several parameters, such as structure, concentration of 1 mM. Then, stock solutions were diluted in cell morphology, crystallinity, and particle size of the end product. media, obtaining a final product concentration of 1, 5, and 10 μM. As mentioned in the Introduction section, reported MOFs 2.5.1. Dark Toxicity Evaluation. The toxicity of the product in the involving H2TPyP as a linker have been obtained using absence of irradiation (from now on, dark toxicity) was evaluated by determining cell viability at two different time points (24 and 72 h techniques where the yield was compromised toward obtaining postincubation) by the Alamar Blue assay. In brief, 24 h after cell high-quality crystals. This work explores the use of green seeding, cells were incubated for 24 h with different concentrations of scCO2 as an efficient solvent for the preparation of new product [0 (control), 1, 5, and 10 μM] previously treated with an H2TPyP-based MOFs at a high yield and in the absence of ultrasonic bath. Later, the cell medium was removed, and cells were large amounts of toxic solvents. For achieving this target, washed four times with Hank’s balanced salt solution (HBSS, hexafluoacetylacetonate metal ligands (M(hfac)2), with typical BioWest) to remove any product remains, followed by fresh medium high solubility in scCO2, were used. In this method, the use of addition. Viability was assessed twice, right after medium addition neutral metallic nodes is preferred due to the limited capacity (viability at 24 h) or after an additional 48 h incubation in standard of scCO2 for dissolving ionic materials. With scCO2 being the culture conditions (viability at 72 h). Three independent experiments were performed for each condition. solvent used in this work for product synthesis, reagent 2.5.2. Photodynamic Treatments. Photodynamic treatments were solubility in this fluid is an important parameter determining performed following the same protocol as for the dark toxicity, but product characteristics. Reagent solubility values in this fluid this time, SKBR-3 cells were incubated with 1 or 5 μM products for 4 were first empirically estimated by a simple visual method h. After 4 h of incubation, the cell medium was removed, and cells carried out in the sapphire windows reactor. Weighed amounts 1082 https://doi.org/10.1021/acs.chemmater.2c03018 Chem. Mater. 2023, 35, 1080−1093 Chemistry of Materials pubs.acs.org/cm Article Figure 1. Plots of the crystal structure of compound [{Co(hfac)2}H2TPyP]n: (a) numbering atoms, (b,c) showing different plane orientations, and (d) stacking. of micronized powder were added to the reactor that was then Initial experiments were carried out lasting 3 h, but after this filled with scCO2 under typical reaction conditions chosen for short period of time, mixtures of end products and unreacted MOF synthesis (e.g., 20 MPa, 60 °C). The turbidity of the H2TPyP were always recovered. Hence, the influence or system, maintained under vigorous stirring, was observed for reaction time was studied by increasing the reaction time in periods of several hours. Only for the H2TPyP linker, the steps, up to a week. The withdrawal of the diffraction peaks of turbidity was permanent, indicating very little solubility in the pure porphyrin linker in the PXRD patterns was chosen as scCO2. In regard to the metal precursors, experimentally, it was the standard indication of the evolution of the H2TPyP toward observed that solubility decreases as follows: Co(hfac)2 > the end product. Upon analyzing the results, after 72 h, no Zn(hfac)2 ∼ Cu(hfac)2 > Ni(hfac)2. The solubility values of further changes in the pattern were observed; thus, the reaction these substances have been compiled in the literature,37 but time was standardized at this time for all of the metals to they are not totally coincident with our observations, likely due complete the reaction. to the different levels of hydration of the reagents. Tests 3.2. Structure of the Synthetized MOFs. Several performed in this work pointed out Ni(hfac)2 having the attempts were performed to synthesize high-quality crystals lowest solubility value compared to the other metal precursors, of all the studied porphyrin-derived MOFs. Used techniques concurrent with its high hydration level. were layering and the solvothermal approach, in which the Since some of the used reagents were not highly soluble in reagents were kept at 120 °C in dimethylformamide in closed scCO2, the process was first optimized by adjusting the vials for 72 h. From all of the performed trials, only the layering synthetic parameters to avoid the presence of large amounts of of Co(II) and H2TPyP in Cl3Et/MeOH resulted in crystals of unreacted molecules after MOF precipitation, which can the necessary quality for performing structural elucidation by contaminate the end product. The first trial consisted in SCXRD. From the rest, only fine particles were precipitated, using a large excess of M(hfac)2 with respect to the organic characterized by PXRD (Figure S1), and not corresponding to linker (molar ratio 6:1). However, no significant changes in the phases precipitated in scCO2. The characteristics of the regard to the structure or purity of the end product were rest of the scCO 2 -precipitated new compounds were observed when comparing this ratio to the stoichiometric determined by comparison with the PXRD pattern of the molar ratio corresponding to the addition of enough metal for Co(II)-derived porphyrin, either prepared by the scCO2 reaching full complexation of all the potential binding sites in method or simulated from the single-crystal structural data. the H2TPyP, for example, the four pyridine groups and the 3.2.1. [{Co(hfac)2}2H2TPyP]n Structure. The porphyrin- inner pyrrole ring. Accordingly, further trials were carried out derived MOF of Co(II) precipitated from layering, with the using a 3.1:1 M(hfac)2/porphyrin molar ratio, aiming to also formula [{Co(hfac)2}2H2TPyP]n, does not show the metal minimize the presence of remaining M(hfac)2 after MOF coordination in the inner porphyrin ring, as shown in Figure precipitation. An exception to this rule was the precipitation of 1a. Crystallographic data is presented in Table S1. The four the MOF of Co(II), for which whatever the experimental pyridines coordinated into metal clusters forming a polymeric conditions, large amounts of unreacted H2TPyP were always structure. This synthesis is believed to be thermodynamically present in the end product. Thus, to precipitate this MOF, the and kinetically favored. The coordination of the pyridines is a CH3Cl cosolvent was employed to enhance linker solubility. highly favorable process because the exocyclic groups are 1083 https://doi.org/10.1021/acs.chemmater.2c03018 Chem. Mater. 2023, 35, 1080−1093 Chemistry of Materials pubs.acs.org/cm Article Figure 2. Plots of the crystal structure of compound [{Co(hfac)2}H2TPyP]n showing the channel voids: (a) a and (b) b directions. highly exposed toward the approach of the metal clusters, and about Co(II) show some deviations from the ideal 90° (values the orbital geometry of the nitrogen is also favorable. In between 85 and 95°), and, besides the pyridyl ring plane, are addition, Co(hfac)2 can coordinate the pyridine without the not perpendicular to the Co−O4 plane 72.27° (Figure 1c). The elimination of any of its hfac moieties, thus maintaining the polymeric sheets are stacked but with some offset. Therefore, ligands and reducing the necessary reaction energy. This the distance between the main planes of adjacent sheets, 4.84 addition process occurs in one step, without the release of any Å, is shorter than the closer Co−Co separation, 6.52 Å (Figure component, and therefore, it can occur in a fast and effective 1d). way. On the contrary, the use of large metallic clusters might The stacking defines channels parallel to the c-axis. The sterically hinder the access of the Co(hfac)2 units to the inner channels have alternative protuberances in the a and b pyrrole ring. Furthermore, the coordination to the pyrrolic ring directions (Figure 2a,b). The estimated accessible volume is would imply the substitution and elimination of the proton in 22 v % per unit cell. Crystals are stable when subjected to the pyrrole (N−H) and the elimination of the hfac moiety as humidity in air, although it is worth noting that it is very hexafluoroacetilcetone upon metal coordination. Overall, the unusual for a crystal with such a high solvent-accessible volume metal coordination of the pyrrolic ring is a highly energetic to be stable when exposed to an open atmosphere. The pore process and therefore less probable to occur in comparison to size was measured along the a-axis, giving a value of 14 Å. the pyridine M(hfac)2 addition. Since the synthesis of the Several weak interactions are established among layers, Co(II) MOF is performed at room temperature, it can be resulting in stable 3D crystals, for example, C−H···F contacts assumed that, in the apparent competing reactions pyridine− that involve the β-carbon pyrrole atom and CF3 groups of metal versus pyrrole−metal, the lack of temperature as energy neighboring layers (H13···F2 2.542 Å), interactions between input is detrimental for the latter versus the former. the H of the pyridine rings and O from the hfac ligand, and [{Co(hfac)2}2H2TPyP]n belongs to the monoclinic crystal weak F···F contacts (Figure 1d). system, with the space group C2/m (12). The asymmetric unit 3.2.2. [M-TPyP]n MOFs Prepared in scCO2. scCO2 was contains one cobalt atom, one hfac ligand, and a quarter of the selected as the reaction medium for synthesizing this type of porphyrin linker. The crystal structure resembles that material as it plays many important roles within MOF synthesis previously described for [{Cu(hfac)2}CuTPyP·6H2O]n,23 in and activation. The solubility of the reagents in different particular on the framework structure, space group, and cell parameters. However, the Co(II) compound in this work solvents guides nucleation and crystal growth kinetics, while shows three key differences: (i) it contains Co(hfac)2 nodes the protic/aprotic character of the solvent determines the instead of Cu(hfac)2 nodes, (ii) the porphyrin is nonmetalated, crystallized phase. scCO2 is an aprotic nonpolar solvent, with a and (iii) the pores do not contain any solvent. The structural strong quadrupole moment, which has a Lewis acid character. analysis revealed a 2D sheet coordination polymer comprising Moreover, it acts as an excellent solvent for neutral and trans-configured Co(hfac)2 nodes, joined by four-connected fluorine-containing metal groups, which makes it an ideal H2TPyP, acting as tetratopic linkers, through the four pyridyl solvent for M(hfac)2 metal clusters. Hence, the role of scCO2 groups. The ensemble defines a (4,4)-net with a rhomboidal in the synthesis of the [M-TPyP]n MOFs is being a solvent for distortion from a regular square grid. The porphyrin ring the metallic node added to the reaction media in the form of system is essentially flat, although it is slightly inclined with M(hfac)2. The solubility of polar compounds in scCO2 has respect to the main sheet planes defined by the Co(II) historically been increased by adding small quantities of liquid positions (angle 5.67°) (Figure 1b). The pyridyl rings are cosolvents to the fluid. However, it was previously demon- 74.07(12)° [(76.55(8)° in the copper compound] tilted strated for ZIF-838 and curcumin29 3D MOFs that these relative to the mean plane of the porphyrin macrocycle. The materials can also be precipitated in scCO2 being only one of coordination sphere about Co(II) is essentially a distorted the reagents, for example, the metal complex, soluble in the elongated octahedron, involving four oxygen donor atoms of fluid. The formation of the [M-TPyP]n MOFs is suggested to the chelating hfac ligands in a plane, with a Co−O bond length proceed in scCO2 by the interaction of dissolved M(hfac)2 of 2.06 Å, and the axial positions occupied by two nitrogen molecules with weakly bonded H2-porphyrin molecules located atoms from pyridyl groups belonging to two different H2TPyP on the surface of the porphyrin, giving place to [(M(hfac)2)x- linkers, with a Co−N bond length of 2.15 Å. The bond angles HyTPyP] complexes and to MOF nuclei. 1084 https://doi.org/10.1021/acs.chemmater.2c03018 Chem. Mater. 2023, 35, 1080−1093 Chemistry of Materials pubs.acs.org/cm Article It is well known that the metal coordination of the pyrrolic ring provides thermodynamic stability to the structure; however, in all of the cases presented here, there was no full coordination of the pyrrolic ring. As previously explained, the formation of [M-TPyP]n MOFs in scCO2 is ruled by the reactivity between the nitrogen atoms of the pyridine and the pyrrole and the metal bonded to hfac. As scCO2 is an aprotic solvent, the free nitrogen would not undergo protonation, and therefore, all the N (the four pyridines and the inner tetrapyrrole ring) are available for bonding in this solvent. The fact that all possible N−M bonds were not completed, even adding an excess of metal, is assumed to be related to the steric hindrance of the M(hfac)2 to enter the porphyrin ring and the lack of enough energy under working conditions to break the M-hfac bond. To test which moiety reacts in preference with the metal, UV−vis titration experiments in CHCl3 were carried out taking the Zn(II) MOF as the case Figure 3. PXRD experimental patterns of [M-TPyP]n MOFs prepared study (Figure S2). For this, a solution of Zn(hfac)2 at different in scCO2 compared to the single-crystal simulated patterns of concentrations was added dropwise to a solution of H2TPyP. [{Cu(hfac) 2 } 2 CuTPyPx6H 2 O] n (Nuwcak) 2 3 and [{Co- In all cases, even the most concentrated one, the four Q bands (hfac)2}2H2TPyP]n. typical of the pyrrole ring remain present, thus indicating the lack of complexation that is exhibited by the declining to only peaks are also present in the scCO2-prepared MOFs of Cu(II) two Q bands. The H2TPyP porphyrin has two different ways of and Zn(II), with a slight shift to lower angles attributed to the binding to metal: (i) the metalation of the central ring, which heterogeneity produced by the coordinated/uncoordinated requires simultaneously both the deprotonation of the two pyrrole ring. Besides, most of the 2θ peaks between 10 and 18° pyrrole groups in the porphyrin, and the substitution of the found for the Co(II) resolved MOF and Cu(II) Nuwcakś were two acetylacetonate ligands in the metal, and (ii) the also present in the different porphyrin samples. In short, for the coordination of the pyridine groups to the metal without the scCO2-synthesized Cu(II) and Zn(II) MOFs, PXRD analysis need for displacing the acetylacetonate ligands. Both reactions indicates similar diffraction peaks, as well as similar relative can enter into the competition. However, it is foreseeable that intensities, which is an indication of predominant isostructural the mechanism of the reaction is multistage and, in any case, features. By contrast, the scCO2-prepared Co(II) and Ni(II) more complex than the bonding of the pyridine groups MOFs can be considered as semiamorphous compounds when necessary to form the two-dimensional network in the MOF. analyzing their PXRD patterns. This is a clear example of a The obtained results in this work indicate that, in scCO2, the MOF, the porphyrin-derived MOF of Co(II), which could be reaction produces a material with a partial degree of prepared as a fully crystalline and semiamorphous material. metalation. Hence, although both reactions compete and The possibility of obtaining pairs of semiamorphous/fully- occur simultaneously, the bonding of the pyridine groups is crystallized MOFs was first described for mesoporous favored, for example, is faster than the metalation. Probably, if carboxylates of Fe(III) involving the MIL-100(Fe) and Fe- a porphyrin is incorporated into the crystal lattice before being BTC pair.24 The semiamorphous product maintains the metalized in the central ring, it is highly unlikely to be crystallographic structure of the crystalline counterpart, as metalized afterward since the metal precursor M(hfac)2 is too well as a significant percentage of the porosity. It should be bulky to diffuse through the pores of the MOF. Another remarked that, for some applications, the semiamorphous argument in favor of this hypothesis is derived from the nature material surpasses the characteristics of the crystalline of the [{Co(hfac)2}2H2TPyP]n compound, obtained by the product.39 In the scCO2 synthesis, semiamorphous MOFs slow diffusion of reagents in organic solvents, and in which the were obtained when using the most [Co(II)] and less [Ni(II)] porphyrin rings are not metalized. This indicates that, under soluble metal reagents. the close-to-equilibrium experimental conditions used for The published structure of the Nuwcak Cu(II) MOF obtaining single crystals, the metallic precursor is favorably showed a fully metal-coordinated (outer and inner ring) consumed to form the crystal lattice bonding to the pyridine product, whereas the Co(II) MOF precipitated in this work groups than to metalize the central ring of the porphyrin. It does not contain any metal ion in the inner pyrrole ring. was found that, even under rapid mixing conditions, and using Nevertheless, both PXRD patterns, simulated from single- an excess of metallic precursor, part of the porphyrin rings has crystal data, were mostly similar in the number of peaks, no option of incorporating metal in the central ring, and the position, and relative intensities (Figure 3). This indicates that four characteristic Q bands of the protonated pyrrole ring are the presence or absence of the metal in the pyrrole ring hardly always observed (Figure S2). causes any changes in the lattice or the crystallographic 3.2.3. Structural PXRD Analysis. The PXRD patterns of the structure. different samples are shown in Figure 3 in the 2θ range of 5 to 3.3. Atomic and Molecular Composition. To further 30°. For comparison, they are contrasted to the simulated ascertain the presence or absence of the metal in the inner ring PXRD pattern of [Co(hfac)2}2H2TPyP]n, resolved in this of the [M-TPyP] n MOFs synthesized in scCO 2 , the work, and to the published structure of [{Cu- composition was measured by performing elemental analysis (hfac)2}2CuTPyP]n (Nuwcak)23 that contained six molecules for C, H, and N atoms and ICP for the metal. Analyzed data of water within the structure. Both, simulated PXRD patterns indicates the precipitation of materials with intermediate show three characteristic peaks at 2θ 5.2, 6.8, and 8.0°. These atomic proportions with respect to the values expected for fully 1085 https://doi.org/10.1021/acs.chemmater.2c03018 Chem. Mater. 2023, 35, 1080−1093 Chemistry of Materials pubs.acs.org/cm Article Figure 4. Scheme of the generic coordination of [M-TPyP]n MOFs. free-base or metalated porphyrin (Table S2). Often, these results do not indicate the precipitation of a mixture of two crystallographic MOF phases, one with the pyrrole coordinated and one noncoordinated, but rather correspond to a single compound with the free-base form of porphyrin and the metalloporphyrin evenly mixed along the structure. Different results were obtained depending on the metal complex used for sample preparation. Considering that all the derived porphyrins underwent exocyclic coordination with M(hfac)2 at the four pyridine moieties, the coordination of the metal inside of the ring could only be partial for Cu(II), Zn(II), and Co(II) MOFs, as it is schematized in Figure 4. For Cu(II) and Zn(II), ca. 30% of the pyrrole rings were coordinated by a metal, while for Co(II), the metalloporphyrin form reached 70% likely due to the high solubility of Co(hfac)2 in scCO2 that favors the Figure 5. FTIR spectra of [M-TPyP]n MOFs compared to pure metalation of the porphyrin. In another situation, for Ni(II), an H2TPyP. excess of metal of ca. 3.5 wt % was measured in the derived porphyrin MOF, even considering full exocyclic and endocyclic pyrrole ring. This band was highly reduced in intensity with coordination. Taking into account the low solubility of respect to free H2TPyP and shifted to higher wavelengths upon Ni(hfac)2 in scCO2, the measured metal excess was attributed complexation with the metal. Besides, a new band emerged to the presence of the end product of the unreacted reagent. corresponding to the N−M bond at ∼1000 cm−1, indicating The FTIR spectra of the synthesized [M-TPyP]n MOFs are that some metal is inserted into the porphyrin ring. shown in Figure 5. All the scCO2 synthesized samples The bands at 1636 and 1200 cm−1 were assigned to C�O displayed similar bands. At high wavenumbers, the signal at of the acetylacetonate in hfac and C�N, respectively. The 3300 cm−1 corresponds to the N−H stretching of the inner bands at 1690−1540 and 1380 cm−1 were assigned to C�C 1086 https://doi.org/10.1021/acs.chemmater.2c03018 Chem. Mater. 2023, 35, 1080−1093 Chemistry of Materials pubs.acs.org/cm Article and C−N stretching vibrations, respectively. The C�N band (Figure 7). For all the samples, the size of the precipitated at 1400 cm−1 of the free pyridine was for the MOFs that particles was in the nano- to low-micrometer range. Cu(II) slightly shifted to higher wavelengths (ca. 15 cm−1). The two bands at ∼1644 and 1600 cm−1 were assigned to ν(CO−) of the ligand, confirming that the metal complex ligand (hfac group) was not released upon assembly of the metal to the pyridine group. The crystalline and semiamorphous Co(II) MOFs present slight differences in the FTIR spectra. Essentially, the signal at 1000 cm−1, corresponding to the N(inner pyrrole ring)−Co bond in the semiamorphous [Co- TPyP]n, is absent in the sample obtained from layering with the formula [{Co(hfac)2}2H2TPyP]n. 3.4. Thermogravimetric Analyses. The thermogravimet- ric decomposition curves of the different MOFs were compared to that of the free H2TPyP (Figure 6). Figure 6. TGA curves of pure H2TPyP and [M-TPyP]n MOFs. H2TPyP decomposition started at an onset temperature of 450 °C, ending at 700 °C, with a two-stage weight loss. The first (19 wt %, 450−475 °C) is attributed to the loss of the peripheral pyridine groups, and the second (25 wt %, 475−540 °C) corresponds to the decomposition of the tetrapyrrole ring. The ratio of these to weight loss values is close to the Figure 7. SEM images of [M-TPyP]n MOFs: (a,b) Cu(II), (c,d) theoretical values of pyridine (50 wt %) and tetrapyrrole (50 Zn(II), (e,f) Co(II), and (g,h) Ni(II). wt %) molecules in the porphyrin. The total weight loss was only of ca. 50 wt % up to 800 °C. The residue has been attributed to the formation of thermally stable carbides. A (Figure 7a,b) and Zn(II) (Figure 7c,d) MOFs precipitated as comparison of the decomposition temperature of pristine crystalline particles of heterogeneous diameters, from nano- to H2TPyP with HxTPyP in the different MOFs reveals that the microsizes, where Cu(II) grew in an elongated way and Zn(II) former has considerably more thermal stability than the in a very well-dispersed form. The hydrodynamic diameter was corresponding metal counterparts. This behavior has been evaluated for the Zn(II) MOF by DLS, giving a mean value of already described for metalated Zn(II), Cu(II), Ni(II), and ca. 140 nm (Figure S3). Crystals obtained from Co(II) (Figure Co(II) tetra(p-carboxylic acid phenyl)porphyrins.6 All MOFs 7e,f) precipitated with a large size as columnar crystals with showed initial weight losses in the range of 125−190 °C, defined edges. attributed to the evaporation of the hfac. This was followed by This is attributed to the enhanced solubility of Co(hfac)2 in several weight loss steps assigned to thermal decomposition, scCO2 when used together with the CHCl3 cosolvent, which varying from metal to metal, which is attributed to the also favored the solubility of the organic linker H2TPyP and, heterogeneity of the network structures. The final residue, thus, the crystal growth. A completely different scenario was varying from 38 wt % in Ni(II) to 25 wt % in Cu(II) MOFs, observed when using Ni(hfac)2 as the metal source. As it was was assigned to the formation of metal oxides. From the assessed, Ni(hfac)2 is the least soluble in scCO2 of all the studied compounds, the gradation of thermal stability was studied metal complexes, which appears to have an effect on Ni(II) > Co(II) > Zn(II) > Cu(II) MOFs. This behavior has the precipitation of the MOF. The Ni(II)-based MOF contains already been described for other porphyrin-related materials numerous nanometric entities (Figure 7g,h). In this case, and is related to the size of the ionic radius of the metal.37 nucleation is clearly favored over crystal growth. This fact is 3.5. Morphological and Textural Properties. The reflected in the precipitation of nanometric (<20 nm) particles, morphology of the precipitated MOFs was observed by SEM resulting in a poorly defined PXRD (Figure 3). To improve the 1087 https://doi.org/10.1021/acs.chemmater.2c03018 Chem. Mater. 2023, 35, 1080−1093 Chemistry of Materials pubs.acs.org/cm Article crystallinity of this MOF, the use of CHCl3 as a cosolvent was powder remained intact, demonstrating that this MOF was attempted, but the final product was not suitable for sufficiently stable to undergo water treatment, at least under characterization as a large amount of pyridine linker, unfeasible the used experimental conditions. Red light with a wavelength to be separated, remained unreacted. Only a few SEM images of 630 nm is generally used for PDT since it corresponds to are reported in the literature showing the morphology of the region of the wavelength used in low-level laser therapy.39 similar compounds obtained in organic solvents. Well-formed Hence, the first step to analyze the suitability of the material structures, namely, microlumps and microprims, have been for PDT studies is to perform UV−vis characterization. The described for the coordination of zinc-10,15,20-tetra(4- intensity and color of porphyrins are derived from the highly pyridyl)porphyrin with Cu(OAc)2 but need the use of conjugated π-electrons, giving characteristic UV−vis spectra cetyltrimethylammonium bromide for adjusting the coordina- that consist of two distinct regions in the near ultraviolet and in tion reaction and improving the crystal growth.40 the visible region. Changes in the conjugation of a particular Textural properties were obtained by measuring N 2 porphyrin would affect the UV−vis absorption spectrum. In adsorption−desorption isotherms at low temperatures (Table this study, the spectra of [Zn-TPyP]n and net H2TPyP were 1). Despite the different compositional and morphological compared (Figure 8). The organic linker (black line) presents Table 1. Textural Properties of the Different Porphyrin MOFs Synthetized in scCO2 sample SBET(m2 g−1) SLangmuir(m2 g−1) Vmp(cm3 g−1) [Cu-TPyP]n 460 490 0.31 [Co-TPyP]n 480 520 0.36 [Zn-TPyP]n 480 520 0.34 [Ni-TPyP]n 480 535 0.32 characteristics measured for the scCO2-precipitated porphyrin- derived MOFs, all of the compounds had a similar N2 adsorption behavior, showing type I isotherms assigned to microporous products (Figure S5). For the samples prepared using scCO2, theoretical surface areas could not be measured due to uncertainties in the structure definition and a large number of defects in the semiamorphous compounds. These samples were evaluated Figure 8. UV−vis absorption spectra of H2TPyP and [Zn-TPyP]n. experimentally. The BET surface area values were consistently derived at the typical porphyrin broad intense Soret band at λmax = 437 ∼480 m2 g−1, with a micropore volume of ∼0.30−0.35 cm3 nm. This band is present at a relatively high value of g−1. This is a clear indication that, independently of the ratio of absorbance since the molecule is mesosubstituted, thus the metalized ring, the textural properties were not corresponding to the transition from the ground state (S0) compromised by this fact. On the contrary, the textural to the second excited state (S2). The second set of less intense properties of the Co(II) MOF obtained from layering could bands are the typical four weak Q bands in the range of 500− not be measured due to the low amount of sample obtained 700 nm with phyllo-type intensities, corresponding to the using this technique. transition S0 → S1 of the free-base porphyrin.41 Upon In this case, the theoretical surface area was calculated from complexation with Zn(II) (yellow line), the Soret band shifted the representation of the crystal structure, taking into account considerably to blue with a λmax = 428 nm, which is attributed the void size. The pore shape was adjusted to a cylinder, using to the loss of symmetry in the porphyrin ring due to an equivalent diameter of 9.1 Å. This led to a theoretical deprotonation of the central pyrrole nitrogen atoms and estimated surface area of ∼1700 m2 g−1. Using this value, the further metalation. In addition, the large complexation of the calculated cell volume corresponds to 814 Å3, equal to the outer pyridyl groups with the metal can also provoke this large crystallographic value. Compared to published data for other shift.42 It has been described that upon full complexation of the H2TPyP MOFs, the only value of surface area found reported porphyrin with the metal in the central ring, the UV−vis was for the compound involving the paddlewheel Cu(II) spectrum also changed with respect to the Q bands since the tetraacetate [Cu2(AcO)4], which was in the order of 800 m2 four bands in H2TPyP corresponding to the NH vibrational g−1.22 excitations get reduced to two due to the increase in the 3.6. Photodynamic Characterization. [Zn-TPyP]n was symmetry in M-TPyP. The spectra of the Zn(II) porphyrin tested in this work as a potential PDT compound because MOF synthesized in scCO2 displayed an intermediate Zn(II) has been reported to be highly biocompatible in situation, in which the four Q bands remained in the spectrum metalloporphyrins.38 but with modified intensities, as it is observed in Figure 8. This 3.6.1. UV−Vis Spectroscopy. UV−vis spectroscopy is often result further indicates that, throughout the [Zn-TPyP]n MOF used to understand porphyrins’ behavior. However, due to the network, the pyrrole ring was only partially metalized. The fact potential lability of the N-metal bond, preliminary stability that the porphyrin MOF preserves the Q bands, and especially studies must be carried out. For this, a sample of the Zn(II) the band at 640 nm, is of extreme importance for its study to porphyrin MOF was dispersed in water by ultrasonication. The use the material as a potential PDT sensitizer. filtered and dried powder was analyzed by PXRD (Figure S4). 3.6.2. Measurement of Singlet Oxygen (1O2) Production. Comparison with untreated samples indicates that the treated To demonstrate that the [Zn-TPyP]n MOF has the potential 1088 https://doi.org/10.1021/acs.chemmater.2c03018 Chem. Mater. 2023, 35, 1080−1093 Chemistry of Materials pubs.acs.org/cm Article to be tested in PDT, an assessment of singlet oxygen dissolved O2 is low.44,45 Nevertheless, aqueous media were production was carried out following two different approaches. used in order to maintain similar conditions to the PDT First, the fluorescence decay caused by the photobleaching of experiment. Despite the relatively low quantum yields, based anthracene-9,10-dipropionic acid in the presence of singlet on the important fluorescence decay, and the well-known oxygen was measured. Second, quantification was performed photosensitizing ability of porphyrins, this experiment by determining singlet oxygen quantum yields, based on the demonstrates that the [Zn-TPyP]n MOF is a compound with phosphorescence of 1O2 at 1270 nm. characteristics adequate to be used in PDT. The fluorescence decay of the ADPA upon irradiation was 3.7. Photodynamic Treatments. 3.7.1. Product Toxicity monitored in time to detect the formation of singlet oxygen in Dark Conditions. Once it was proven by UV−vis that [Zn- (Figure 9). The same light source, operating in the same TPyP]n can be used in PDT, the toxic effects of the product on cell viability, in the absence of light irradiation (dark toxicity), were evaluated using the Alamar Blue assay at two different time points as the amount of fluorescence produced is proportional to the number of living cells. As can be seen in Figure 10, none of the concentrations tested resulted in cytotoxicity after 24 h postincubation, but after 72 h of incubation, cell viability was significantly lower with 5 (78.6%) and 10 μM (74.1%) concentrations. Toxicity was concen- tration-dependent; the higher the concentration, the lower the viability. 3.7.2. Cell Viability after Photodynamic Treatments. Photodynamic treatments were performed with 1 and 5 μM concentrations of products because higher concentrations resulted in toxicity at 72 h postincubation in dark conditions. Figure 9. Fluorescence emission spectra of ADPA after irradiating for 15 min at 630 nm in the presence of the [Zn-TPyP]n photosensitizer. Photodynamic treatments were executed in an irradiator dispositive that homogeneously irradiates all the cells. Irradiation was performed at room temperature, which can conditions (630 nm, 55 mW cm−2 intensity) as in the PDT affect cell viability. For this reason, dark toxicity was also tested measurements, was used for irradiation. The wavelength of the under these conditions. To confirm that phototoxicity was due irradiation closely corresponds to the typical Q-band of the to product internalization, the product was incubated for only porphyrin, nearest to the red region. An important decrease in 4 h, followed by an exhaustive wash to eliminate all the the fluorescence intensity was detected already after 5 min, noninternalized product (Figure 11); then, cells were showing the photobleaching of the ADPA to the non- fluorescent endoperoxide43 in the presence of the irradiated irradiated for 15 min. photosensitizer. After 15 min, no fluorescence signal was The results obtained in dark toxicity under these conditions detected, and the ADPA was irreversibly quenched. This result were similar to that obtained in the first experiment; that is, the confirms the potential of [Zn-TPyP]n MOF to be used as a product was not toxic at any time point tested, except for 5 μM photosensitizer in PDT treatment. product at 72 h after product incubation (78.6%). After 15 min In a second set of experiments, the generation of singlet of irradiation, when incubating with 5 μM product, cells oxygen was performed by the direct measurement of its well- showed a significant decrease in cell survival after both 24 and known phosphorescence at 1270 nm. Perinaphtenone was used 72 h (69.9 and 35.7% viability, respectively). On the other as the standard reference in order to quantify this process. As hand, incubation with 1 μM product only led to a significant expected, similar values for the dispersion of pure porphyrin decrease in cell survival after 72 h (37.2% viability). These crystals (ϕΔ = 2.7%) and the Zn(II) MOF (ϕΔ = 2.3%) was results are in agreement with that obtained previously using the detected, although in the first case, the large H2TPyP crystals same SKBR-3 cell line and a porphyrin linked to Zn(II) metal do not disperse homogeneously in the solvent, unlike the case complex (Na-ZnTCPP);46 for example, using 1 μM of this of the MOF. These values were found to be quite low, which is product, cell viability was reduced to more than a half. Other the result expected in aqueous dispersions where, in authors have also reported a reduction in cell viability using comparison with other solvents, the concentration of the water-soluble porphyrins linked to the Zn(II) metal complex.47 Figure 10. Dark toxicity: cell viability of SKBR-3 cells incubated for 24 h in dark conditions with different concentrations of the product (1, 5, and 10 μM) and a control (0 mM) at 24 and 72 h. * indicate statistically significant differences in cell viability compared to the control at each time point. Experiments were done in triplicate. 1089 https://doi.org/10.1021/acs.chemmater.2c03018 Chem. Mater. 2023, 35, 1080−1093 Chemistry of Materials pubs.acs.org/cm Article Figure 11. Photodynamic treatment effects after 15 min of irradiation. Cell viability was determined by Alamar Blue for SKBR-3 cells incubated without (control) or with 1 or 5 μM product for 4 h followed by cell wash. Cell viability was determined after incubation (24 h) either in dark conditions (not irradiated) or after 15 min of irradiation at λex 620−630 nm (irradiated) and at 72 h. Three independent experiments were performed for each set of conditions. * indicate statistically significant differences in the cell viability between the control and product at each time point and condition. Experiments were done in triplicate. Figure 12. Live SKBR-3 cells incubated with 1 μM product for 4 h (a,b) and 24 h (c,d) and observed under a confocal microscope. To analyze the localization of the product, fluorescence mode was used. Product fluorescence emission was detected in the range of λ 600−763 nm (orange) by exciting the cells using a λ 405 nm laser (15% of the laser power). WGA fluorescence emission (membrane) was detected in the range of λ 500− 550 nm (green) by exciting the cells using a λ 488 nm laser (12 1% of the laser power). Maximum projection (a,c) and orthogonal projection of z- stacks (b,d). Arrows point to some aggregates where the cross is positioned (b,d). Scale bar 20 μm. 3.8. Internalization of the Photosensitizers. Internal- were visible in the orthogonal projection (Figure 12b, arrows). ization of the product was analyzed by confocal microscopy. However, after 24 h of incubation, product internalization was SKBR-3 cells were incubated with 1 μM product for 4 and 24 clearly observed in both images, maximum (Figure 12c) and h, washed four times to eliminate all the noninternalized orthogonal (Figure 12d) projections. Nevertheless, 4 h of products, and incubated with WGA-488 for 15 min to visualize incubation was enough to kill more than 50% of the cell the plasma membrane (limit of the cell). Consecutive optical population after 15 min of irradiation. slices throughout the cell volume were taken to obtain the 2D Considering that [Zn-TPyP]n has been shown to generate 1 maximum projection (Figure 12a,c), whereas orthogonal O2 and that confocal studies have shown that [Zn-TPyP]n sections allowed to locate the product within the limits of could be effectively internalized by cells, it is assumed that the the cell (Figure 12b,d). After 4 h of incubation, the product reduced cell viability during light irradiation is due to the could be detected as a red background inside every single cell generation of ROS. [Zn-TPyP]n exhibits high phototoxicity in the maximum projection (Figure 12a), but only aggregates and low dark toxicity at a concentration of 1 μM. This 1090 https://doi.org/10.1021/acs.chemmater.2c03018 Chem. Mater. 2023, 35, 1080−1093 Chemistry of Materials pubs.acs.org/cm Article photosensitizer is highly efficient in inducing cell death, more Authors than 62% after 72 h of a tumorigenic human mammary Márta Kubovics − Institute of Materials Science of Barcelona epithelial cell line. Other authors have demonstrated the use of (ICMAB-CSIC), 08193 Bellaterra, Spain; orcid.org/ different photosensitizers for PDT as an anticancer ther- 0000-0002-2441-8562 apy.46−49 Oriol Careta − Department de Biologia Cel·lular, Fisiologia i Immunologia, Universtitat Autònoma de Barcelona (UAB), 4. CONCLUSIONS 08193 Bellaterra, Spain Oriol Vallcorba − ALBA Synchrotron Light Source, 08290 scCO2 proved to be a rapid, green, and effective approach for Cerdanyola del Vallés, Spain; orcid.org/0000-0001- preparing MOFs from highly insoluble organic ligands. In this 6499-7688 way, a collection of [M-TPyP]n [M = Cu(II), Zn(II), Co(II), Guillermo Romo-Islas − Department of Inorganic and and Ni(II)] MOFs was prepared using scCO2 as reaction Organic Chemistry, Barcelona University, 08028 Barcelona, media using mild conditions. Elemental analysis, PXRD Spain; Institute of Nanoscience and Nanotechnology patterns, FTIR, and UV−vis characterizations indicated that (IN2UB), Barcelona University, 08028 Barcelona, Spain; MOFs obtained from Cu(II), Zn(II), Ni(II), and Co(II) were orcid.org/0000-0002-2841-9546 obtained as porous networks, where the full coordination of all Laura Rodríguez − Department of Inorganic and Organic binding sites was not complete. In addition, [{Co(hfac)2}- Chemistry, Barcelona University, 08028 Barcelona, Spain; 2H2TPyP]n was obtained from layering, whose structure was Institute of Nanoscience and Nanotechnology (IN2UB), elucidated from SCXRD. This MOF was characterized for Barcelona University, 08028 Barcelona, Spain; orcid.org/ having a porous network from which the inner pyrrole ring 0000-0003-1289-1587 remained uncoordinated. From all of the MOFs obtained, that Jose A. Ayllón − Department de Química, Universtitat of Zn(II) was used for PDT. The uncompleted coordination of Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain; the inner pyrrole ring gave place to the presence of a Q-band at orcid.org/0000-0001-7965-7424 640 nm, which was key for targeting the MOF as a potential Complete contact information is available at: photosensitizer. https://pubs.acs.org/10.1021/acs.chemmater.2c03018 ■ * ASSOCIATED CONTENT sı Supporting Information Author Contributions The manuscript was written through the contributions of all authors. All authors have given approval to the final version of The Supporting Information is available free of charge at the manuscript. https://pubs.acs.org/doi/10.1021/acs.chemmater.2c03018. Funding Reaction products from the conventional synthesis of This work was supported by the Spanish Ministry of Science MOFs; UV−vis spectra of complexation experiments of and Innovation MICINN through the Severo Ochoa Program pure porphyrin with Zn(II); crystal data of [{Co- for Centres of Excellence (CEX2019-000917-S) and the (hfac)2}H2TPyP]n; particle size of [Zn-TPyP]n; PXRD Spanish National Plan of Research with projects PID2020- confirming the stability of [Zn-TPyP]n; BETs of scCO2 115631GB-I00 (C.D., A.M.L.-P., and M.K.), PID2020- obtained MOFs; and formulas and elemental analyses of 116844RB-C21, and 2017SGR503-Generalitat de Catalunya MOFs (PDF) (C.N. and O.C) and PID2019-104121GB-I00 (G. R.-I. and L.R.). Accession Codes Notes CCDC 2190259 contains the supplementary crystallographic The authors declare no competing financial interest. data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_request@ccdc.cam.ac.uk, or by contacting The Cam- ■ ACKNOWLEDGMENTS M.K. acknowledges the financial support from the European bridge Crystallographic Data Centre, 12 Union Road, Union’s Horizon 2020 research and innovation programme Cambridge CB2 1EZ, UK; fax:+44 1223 336033. under the Marie Sklodowska-Curie Cofund grant (agreement no. MSCA-COFUND-DP/0320−754397). C.N. would like to ■ AUTHOR INFORMATION Corresponding Authors thank the staff from the Servei de Microscòpia of Universitat Autònoma de Barcelona. A.M.L.P. would like to thank the staff from the Servei of XRD at the ICMAB and ALBA synchrotron Concepción Domingo − Institute of Materials Science of for the beamtime. This work has been done in the framework Barcelona (ICMAB-CSIC), 08193 Bellaterra, Spain; of the doctoral program “Chemistry” of the Universitat orcid.org/0000-0002-6976-8283; Email: conchi@ Autònoma de Barcelona by M.K. The authors also acknowl- icmab.es edge the CSIC-PTI platform SUSPLAST. Carme Nogués − Department de Biologia Cel·lular, Fisiologia i Immunologia, Universtitat Autònoma de Barcelona (UAB), 08193 Bellaterra, Spain; orcid.org/0000-0002-6361- 8559; Email: carme.nogues@uab.cat ■ REFERENCES (1) Liu, J.; Zhou, W.; Liu, J.; Howard, I.; Kilibarda, G.; Schlabach, S.; Coupry, D.; Addicoat, M.; Yoneda, S.; Tsutsui, Y.; Sakurai, T.; Seki, Ana M. López-Periago − Institute of Materials Science of S.; Wang, Z.; Lindemann, P.; Redel, E.; Heine, T.; Wöll, C. Barcelona (ICMAB-CSIC), 08193 Bellaterra, Spain; Photoinduced charge-carrier generation in epitaxial MOF thin films: orcid.org/0000-0002-3777-3205; Email: amlopez@ High efficiency as a result of an indirect electronic band gap? 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