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Cite This: ACS Appl. Mater. Interfaces 2018, 10, 8527−8535

Designing Porphyrinic Covalent Organic Frameworks for the Photodynamic Inactivation of Bacteria Jan Hynek,†,‡ Jaroslav Zelenka,$ Jiří Rathouský,# Pavel Kubát,# Tomás ̌ Ruml,$ Jan Demel,† and Kamil Lang*,† Institute of Inorganic Chemistry, Czech Academy of Sciences, Husinec-Ř ež 1001, 250 68 Ř ež, Czech Republic Department of Inorganic Chemistry, Faculty of Science, Charles University, Hlavova 2030, 128 43 Praha 2, Czech Republic $ Department of Biochemistry and Microbiology, University of Chemistry and Technology Prague, Technická 5, 166 28 Praha 6, Czech Republic # J. Heyrovský Institute of Physical Chemistry, Czech Academy of Sciences, Dolejškova 3, 182 23 Praha 8, Czech Republic † ‡

S Supporting Information *

ABSTRACT: Microbial colonization of biomedical devices is a recognized complication contributing to healthcare-associated infections. One of the possible approaches to prevent surfaces from the biofilm formation is antimicrobial photodynamic inactivation based on the cytotoxic effect of singlet oxygen, O2(1Δg), a short-lived, highly oxidative species, produced by energy transfer between excited photosensitizers and molecular oxygen. We synthesized porphyrin-based covalent organic frameworks (COFs) by Schiff-base chemistry. These novel COFs have a three-dimensional, diamond-like structure. The detailed analysis of their photophysical and photochemical properties shows that the COFs effectively produce O2(1Δg) under visible light irradiation, and especially three-dimensional structures have strong antibacterial effects toward Pseudomonas aeruginosa and Enterococcus faecalis biofilms. The COFs exhibit high photostability and broad spectral efficiency. Hence, the porphyrinic COFs are suitable candidates for the design of antibacterial coating for indoor applications. KEYWORDS: covalent organic framework, porphyrin, singlet oxygen, photodynamic, biofilm, antibacterial coating

1. INTRODUCTION Increasing resistance of bacteria to antibiotics makes curing many diseases difficult.1 In addition, bacteria can form cohesive networks, called biofilms, held together by a hydrated extracellular polymeric substance, consisting of proteins, lipids, polysaccharides, and nucleic acids. This form of bacterial growth is prevalent in nature and allows metabolic symbiosis between different bacterial strains or differently supplied bacteria from one strain. The close proximity of bacterial cells and the presence of extracellular DNA in the biofilm matrix enable transfer of genetic elements encoding virulence and resistance factors. The biofilms of virulent and multiresistant bacteria can be several orders of magnitude more resistant to antibiotics and disinfectants than separated bacteria; this represents a serious problem, especially in hospitals with local accumulation of immunocompromised persons susceptible to © 2018 American Chemical Society

infections. One of the bacteria, which effectively forms resistant biofilms causing severe problems in the food industry and medicine, is Pseudomonas aeruginosa.2 To mitigate biofilm formation, numerous antibacterial agents and antiseptic techniques have been proposed.3 A promising method for fighting microorganisms is antimicrobial photodynamic inactivation (PDI).4 It relies on the formation of reactive oxygen species (ROS), including singlet oxygen, O2(1Δg), a short-lived, highly oxidative agent with bactericidal5 and virucidal6 properties generated in situ via energy transfer from an excited molecule of a photosensitizer to an oxygen molecule. Typical examples of photosensitizers are porphyrins Received: December 30, 2017 Accepted: February 22, 2018 Published: February 22, 2018 8527

DOI: 10.1021/acsami.7b19835 ACS Appl. Mater. Interfaces 2018, 10, 8527−8535

Research Article

ACS Applied Materials & Interfaces Scheme 1. Syntheses of Precursors and COFs Including Abbreviations Used in the Text

and phthalocyanines.7 However, these molecules tend to aggregate which results in losses of the photosensitizing activity. A possible solution to overcome these obstacles is the synthesis of porphyrin-based solid photosensitizers with a forced and well-defined arrangement of porphyrin molecules. To name several of these solids, layered metal hydroxides,8 metal−organic frameworks,9−11 conjugated microporous polymers,12 or covalent organic frameworks13 have been already described, including their photoactivity with respect to the production of O2(1Δg).

Covalent organic frameworks (COFs) are a class of porous crystalline materials composed of organic building blocks connected by reversibly formed covalent bonds, such as boroxine,14 boronyl ester,15 triazine,16 or imine17 linkage. Since 2005, when the first COFs were published by Yaghi et al.,18 various two-dimensional (2D) frameworks have been synthesized.19 In contrast, only a limited number of threedimensional (3D) frameworks has been reported so far.20,21 Although COFs have been first explored for gas adsorption and storage applications, the incorporation of functional building blocks has opened up new potential applications. Especially, 8528


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ACS Applied Materials & Interfaces

detection module in a time-correlated single-photon counting regime. The decay curves were fitted with exponential functions using the iterative reconvolution procedure of the DAS6 software (v. 6.8, Horiba Jobin Yvon). Transient absorption measurements were performed on a laser kinetic spectrometer LKS 20 (Applied Photophysics, U.K.) equipped with a Lambda Physik FL 3002 dye laser (425 nm, pulse width 28 ns), a 150 W Xe lamp, and a R928 photomultiplier (Hamamatsu). The triplet state kinetics was monitored either by transient absorption at 460 nm (3D-TPP, 2D-TPP) or phosphorescence at 710 nm (3DPdTPP). The bimolecular rate constants kO2 for the quenching of the triplet states by molecular oxygen were evaluated using the Stern− Volmer equation, 1/τT = 1/τT0 + kO2 [O2], and oxygen solubility in air-saturated acetonitrile (2.42 mM),32 where τT and τT0 are the triplet state lifetimes at given oxygen concentration [O2] and under oxygenfree conditions, respectively. The fractions of the porphyrin triplet states quenched by molecular oxygen in air-saturated dispersions were air 1 calculated as f air T = 1− τT /τT0. The kinetics of O2( Δg) luminescence was recorded at 1270 nm at the right angle to 425 nm laser pulses (FL3002 dye laser, pulse width ∼28 ns) using a homemade Ge detector. The signal from the detector was collected in a 600 MHz oscilloscope (Agilent Infiniium), and the signal-to-noise ratio was improved by the averaging of 500 individual traces. The quantum yields of singlet oxygen formation (ΦΔ) in acetonitrile were estimated by the comparative method using TPP (ΦΔ = 0.60)33 as a standard. 2.3. Syntheses of Precursors and COFs. The porphyrin building blocks were synthesized as shown in Scheme 1 (see the Supporting Information for details). The aldehyde derivative of 5,10,15,20tetraphenyl porphyrin 1 was obtained by the Suzuki−Miyaura crosscoupling reaction and the following metalation of 1 with Pd(acac)2 in toluene yielded 2 (Supporting Information (SI) Figures S1−S4). 2.3.1. Preparation of 3D-TPP. Compound 1 (50 mg, 75 μmol) was dissolved in 2.7 mL of 1,4-dioxane and 14.2 mg of tetrakis(4aminophenyl)methane (37 μmol) was dissolved in 0.8 mL of 1,4dioxane. Then both solutions were mixed together in a glass ampule and 0.2 mL of 3 M acetic acid was added. The ampule was treated by three freeze−pump−thaw cycles, evacuated, sealed up, and heated up to 120 °C for 72 h. After cooling down, the ampule was opened and the resulting precipitate was collected by filtration. The product was properly washed by 1,4-dioxane and THF, followed by 24 h Soxhlet extraction with THF. Yield: 48 mg, 78%. Elemental Analysis Calcd: C, 85.38%; H, 4.41%; N, 10.21%. Found: C, 81.35%; H, 4.43%; N, 9.24%. 2.3.2. Preparation of 3D-PdTPP. Compound 2 (58 mg, 75 μmol) was dissolved in 2.7 mL of 1,4-dioxane, and 14.2 mg of tetrakis(4aminophenyl)methane (37 μmol) was dissolved in 0.8 mL of 1,4dioxane. Both solutions were inserted into a glass ampule, and 0.2 mL of 3 M acetic acid was added. The ampule was treated by three freeze− pump−thaw cycles, evacuated, sealed up, and heated up to 120 °C for 72 h. After cooling down, the ampule was opened and the resulting precipitate was collected by filtration. The product was properly washed by 1,4-dioxane and THF, followed by 24 h Soxhlet extraction with THF. Yield: 47 mg, 67%. Elemental Analysis Calcd: C, 75.77%; H, 3.70%; N, 9.06%. Found: C, 70.99%; H, 3.91%; N, 8.19%. 2.3.3. Preparation of 2D-TPP (COF-366). The material was prepared according to the procedure reported by Yaghi et al.31 5,10,15,20-Tetrakis(4-aminophenyl)porphyrin (54 mg, 80 μmol) was dissolved in 2 mL of ethanol, and 22.4 mg of terephthalaldehyde (167 μmol) was dissolved in 2 mL of mesitylene. Both solutions were inserted into a glass ampule, and 0.4 mL of 6 M acetic acid was added. The ampule was treated with three freeze−pump−thaw cycles, evacuated, sealed up, and heated up to 120 °C for 72 h. After cooling down, the ampule was opened and the resulting precipitate was collected by filtration. The product was properly washed by 1,4dioxane and THF, followed by 24 h Soxhlet extraction with THF. Yield: 42 mg, 58%. Elemental Analysis Calcd: C, 82.74%; H, 4.40%; N, 12.86%. Found: C, 76.84%; H, 4.80%; N, 12.85%. 2.4. Photosensitization Activity. The photosensitizing ability of COFs was assessed using the reaction of photoproduced O2(1Δg) with 9,10-diphenyl anthracene leading to the corresponding endoperoxide. The course of the reaction was followed by a decreasing absorption of

porphyrins are promising building blocks due to their planar geometry and rigidity coupled with inherent functionalities such as photosensitizing and redox-active properties. These properties enabled to use porphyrinic COFs in catalytic,22−25 electrocatalytic,26 sensing,27 photochemical,13,28 energy harvesting,29 and storage30 applications. We present the syntheses and characterization of porphyrinic COFs where the porphyrin blocks are linked together by the Schiff-base reaction. The sp3 hybridized carbon atom in tetraphenyl methane connected with the planar porphyrin linker leads to the diamond-like framework. The results show that the COF topology, i.e., 2D (COF-366)31 vs 3D structures, affects the productivity of O2 ( 1Δ g ) and the resulting antibacterial properties. Especially, the 3D COF materials exhibit excellent antibacterial effects under visible light and are supposed to be well-suited for the fabrication of antibacterial polystyrene coatings due to high photostability, broad spectral efficiency, and good dispersibility in polystyrene.

2. EXPERIMENTAL SECTION 2.1. Materials. Tetrakis(4-aminophenyl)methane (Manchester Organics, UK), Pd(PPh3)4, Pd(acac)2, MgSO4, terephthalaldehyde, polystyrene (average MW ∼192000 g mol−1), (aminomethyl)polystyrene (200−400 mesh, extent of labeling: 4.0 mmol g−1 loading, 2% cross-linked), 5,10,15,20-tetraphenylporphyrin (TPP), acetonitrile (Chromasolv), and 9,10-diphenylanthracene (all Sigma-Aldrich), 5,10,15,20-tetrakis(4-aminophenyl)porphyrin, 4-formylbenzeneboronic acid (both Frontier-Scientific, USA), 5,15-dibromo-10,20diphenylporphyrin (DPPBr, PorphyChem, France), CH2Cl2 (Lachner, Czech Republic), NaCl, NaHCO3, and K2CO3 (all Lachema, Czech Republic), tetrahydrofuran (THF), 1,4-dioxane (both Penta, Czech Republic), and Hoechst 33342 (ThermoFisher Scientific) were used as purchased. For Suzuki coupling reactions, 1,4-dioxane (water-free, VWR Chemicals) was dried using an SP-1 solvent purification system (LC Technology Solutions). 2.2. Instrumental Methods. 1H and 13C NMR spectroscopy was performed on a Varian Mercury 400Plus Instrument or a JEOL 600 MHz NMR spectrometer. 13C cross-polarization NMR spectra were recorded using a JEOL 600 MHz NMR spectrometer at 10 kHz MAS rate. Powder X-ray diffraction patterns were recorded on a PANalytical X’Pert PRO diffractometer in the Bragg−Brentano geometry equipped with a conventional X-ray tube (Co Kα, 40 kV, 30 mA) and a multichannel detector X’Celarator with an antiscatter shield. Thermal analyses were carried out using a Setaram SETSYS Evolution-16-MS instrument coupled with a mass spectrometer. Fourier transform infrared spectra (FTIR) were collected on a Nicolet NEXUS 670-FT spectrometer in KBr pellets (2000−400 cm−1) or using a Praying Mantis (Harrick) diffuse reflection accessory (4000−2000 cm−1). CHN elemental analysis was performed using a standard combustion technique. Textural properties were determined by the analysis of nitrogen sorption isotherms obtained at ca. 77 K with a Micromeritics ASAP 2010 apparatus. Prior to sorption experiments, the samples were outgassed at 80 °C for at least 24 h. The sorption isotherms were analyzed by several techniques, including the BET method, Broekhoff−de Boer (BdB) t-plots, and NLDFT methods for various pore shapes and interaction potentials (as provided by the Micromeritics software), in order to achieve consistent pore sizes and surface areas. The UV/vis absorption spectra of COF dispersions in acetonitrile were recorded on a PerkinElmer Lambda 35 equipped with a Labsphere RSA-PE-20 integration sphere. The luminescence properties of COFs and O2(1Δg) luminescence spectra were monitored on a Fluorolog 3 spectrometer using a cooled TBX-05-C photon detection module (Horiba Jobin Yvon) and a Hamamatsu H10330-45 photomultiplier, respectively. The fluorescence lifetime measurements were performed using a laser diode excitation at 405 nm (NanoLED405LH, pulse width 750 ps, repetition rate 1 MHz). The fluorescence was recorded at 660 and 720 nm using a cooled TBX-05-C photon 8529


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ACS Applied Materials & Interfaces Table 1. Texture Parameters of Investigated COFsa COF

texture

SBET (m2 g−1)

Vmicrob (cm3 g−1)

Smicrob (m2 g−1)

Vmesob (cm3 g−1)

Smesob (m2 g−1)

Dmax (nm)

Sext (m2 g−1)

3D-TPP 3D-PdTPP 2D-TPP

micro-meso bimodal mesoporous microporous

83 50 475

0.02 0 0.20

43 0 475

0.08 0.12 0

35 50 0

1.2, 9 7−8 ∼2

5 very small very small

a

SBET stands for the BET surface area; V is the pore volume of micropores or mesopores determined by the Broekhoff−de Boer t-plot method; Dmax is the pore width determined by the NLDFT method, corresponding to the maximum/maxima of the pore size distribution; Sext is the external surface area. bDetermined by the Broekhoff−de Boer t-plot method. 9,10-diphenyl anthracene between 230−410 nm using UV/vis spectroscopy. In each experiment, 1.0 mg of COF was dispersed in 20 mL of 10−4 M 9,10-diphenyl anthracene in acetonitrile and placed in a quartz cuvette. After sonication for 10 s, the dispersion was stirred with a magnetic bar and continuously irradiated by a 300 W Xe lamp (ozone free, Newport) equipped with a water filter and a long-pass filter (435 nm, Newport) for 3 h. The aliquots were taken at regular time intervals, centrifuged (10000 rpm, 10 min, Hettich Rotina 35 centrifuge), filtered through a 0.2 μm PTFE Millipore filter, and inserted into a 10 × 10 mm quartz cell to measure the corresponding UV/vis spectrum. The irradiation experiments were performed in air. 2.5. Preparation of Antibacterial Coatings. The antibacterial coatings were prepared on 12 × 12 mm glass plates. One mg mL−1 of polystyrene was dissolved in saturated THF solution of (aminomethyl)polystyrene (∼0.15 mg mL−1), and 60 μL of this solution was drop-casted on the plate. Then, fine powder of COFs was homogeneously scattered on the surface of the wet coating (∼0.35 mg cm−2). The coatings for blank experiments were prepared by the same procedure; however, no COF was added. The solvent was evaporated to dryness in air, and the plates were thoroughly washed with water and air-dried. The coatings were tested for their stability as follows: the coatings were immersed in water and continually irradiated by 460 nm light in the same setup as used for antibacterial tests. The aliquots taken in regular intervals up to 24 h were analyzed for the presence of dissolved porphyrins by UV/vis spectroscopy. 2.6. Bacterial Inactivation Testing. Two bacterial strains, Gramnegative Pseudomonas aeruginosa and Gram-positive Enterococcus faecalis were from the microbial collection of the University of Chemistry and Technology, Prague. The bacteria cultures were maintained in Luria−Bertani (LB) agar and cultured in the LB medium, pH 7. For viability experiments, the 24 h bacterial cultures were inoculated into Petri dishes with the fresh LB medium to a final density of 1 McFarland. Then the coated plates were immersed into the media with the functional side downward. The dishes were either stored in the dark (blank experiments) or irradiated either with a 150 W halogen lamp (Conrad electronics) equipped with a water filter (50 mW cm−2) or with 12 × 10 W LED light source (Cameo) at 460 (20 mW cm−2) or 525 (7 mW cm−2) nm for the indicated time period (24 h, 48 h). Next, the plates were removed from the dishes, washed three times with phosphate-buffered saline, and the formation of bacterial biofilms was inspected with a bright field inverted microscope with 400× magnification. The biofilms were then fixed with 100% ice-cold methanol for 15 min and stained with the Hoechst stain according to the manufacturer protocol.34 The stained glasses were put with the functional side downward onto the dishes with a microscopic cover glass bottom (MatTek) and inspected using a confocal microscope (Olympus/Andor xD, 400× magnitude). The Hoechst staining was measured using 405 nm laser excitation. Images were analyzed using the ImageJ software, and the biofilm density was measured as the total stained surface of the image. The data sets were analyzed with the SigmaStat software (Systat). The Student t test was performed after testing for the normal distribution. The results with p < 0.01 were considered significant due to multiple comparisons.

jugated microporous polymers are efficient photosensitizers of O2(1Δg).12 The utilization of this functionality and to impose adhesion of bacteria on the coating surfaces, we designed COFs bound together by the Schiff-base reaction. In this design, the terminal −NH2 groups provide stronger adhesion with bacterial cell walls.35 2D-TPP, also known as COF-366, was prepared using a procedure based on the Schiff-base formation.31 Threedimensional COFs were synthesized analogously to the method developed by Yaghi et al., who prepared a CN bonded COF with the diamond-like structure from tetrakis(4-aminophenyl)methane and terephthaldehyde (Scheme 1).20 Instead of terephthaldehyde, we used porphyrinic linker molecules 1 and 2 with the ability to produce O2(1Δg). At the final step, the resulting solids were purified by the Soxhlet extraction with THF to remove unreacted components. The COF materials were fully characterized by 13C MAS NMR, FTIR, powder Xray diffraction, and thermal and elemental analyses (details in the Supporting Information). The textural properties were assessed by the analysis of N2 adsorption isotherms. Powder X-ray diffraction pattern (SI Figure S5) of 2D-TPP shows characteristic diffractions at 4.0° and 9.1°, corresponding to a basal spacing of 25.2 Å, which is in agreement with the reported values for COF-366.31 In contrast, both 3D-TPP and 3D-PdTPP do not have any characteristic diffractions, confirming their rather disordered nature. 13C CP-MAS NMR spectra display broad signals in the range between 110 and 138 ppm (SI Figure S6). The peaks at 158 ppm indicate the presence of imine linkages of the building blocks. In the case of 2D-TPP, the peak of terminal aldehyde groups at 190 ppm can be also recognized. Three-dimensional COFs show intensive peaks of aromatic carbon atoms of the tetraphenyl methane building blocks at 142 ppm and small peaks of quaternary carbon atoms of tetraphenyl methane at 65 ppm. FTIR spectra (SI Figure S7) exhibit the CN stretching vibration at 1620 cm−1, which is in accordance with the formed imine bond in prepared COFs. The stretching aromatic C−H vibrations are located in the region between 3024 and 3055 cm−1, and a sharp pyrrole ring vibration is at 794−798 cm−1. The peaks at 1700 cm−1 belong to residual aldehyde groups, and the peaks at 3380 and 3470 cm−1 are attributed to stretching N−H vibrations of residual NH2 groups. A characteristic signal of the pyrrole N−H stretching at 3315 cm−1 disappears in the case of 3D-PdTPP because of the exchange of hydrogen atoms for coordinated palladium atom. The combination of FTIR and 13C CP-MAS NMR data confirm the proposed structure of 3D COFs. The thermal analyses reveal thermal stability of porphyrinic COFs up to 300 °C in air (SI Figure S8). The combustion process is accompanied by the evolution of CO2, H2O, and NOx. The porosity of COFs was determined by the analysis of the nitrogen sorption isotherms at ca. 77 K (Table 1). The sorption isotherms of all COFs feature very broad hysteresis loops with the desorption branch running in parallel to the adsorption one,

3. RESULTS AND DISCUSSION 3.1. Synthesis and Structural Characterization. We have already demonstrated that porphyrin-containing con8530


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phosphorescent, the triplet states of 3D-PdTPP can be probed by luminescence spectroscopy (Table 2). In the absence of

joining it at the closure point at the relative pressure as low as ca. 0.01 (SI Figure S9). As nitrogen desorption at such low relative pressures is a very slow process, it requires as much as 10 h to achieve the equilibrium. It is apparently due to an elastic nature of the polymer network leading to structural expansion caused by the breakthrough of nitrogen molecules during adsorption and their retention or blocking during desorption. As 3D-TPP contains both micropores and mesopores, the range of validity of the Brunauer−Emmett−Teller (BET) equation is limited to relative pressures between 0.05 and 0.12. The total surface area is significantly lower than that for other COF materials,31 probably due to interpenetration of the diamond-like structure. The complexation of palladium in 3DPdTPP led to a disappearance of micropores and to a decrease in the BET surface area when compared with 3D-TPP. The pore size distribution of mesopores is wide without any characteristic width, which indicates that these mesopores are rather related to structural defects than to organized mesoporosity. The 2D-TPP material contains wider micropores about 2 nm in size, which is in an agreement with the structure where the distance between the carbon centers of the opposite phenyl rings is approximately 2 nm. As the pore width is at the border between micropore and mesopore range, the BET model was applicable providing a surface area of 475 m2 g−1 and a pore volume of 0.20 cm3 g−1. These values are lower than those reported for COF-366 (735 m2 g−1 and 0.32 cm3 g−1).31 3.2. Spectral and Photochemical Properties. The spectral features of COFs and the triplet state dynamics are fundamental properties for assessment of materials designed to produce O2(1Δg). The UV/vis absorption spectra of the COF dispersions in acetonitrile display the characteristic Soret and Q bands of the porphyrin units (Figure 1). In comparison with

Table 2. Photophysical Properties of COF Dispersions in Acetonitrilea O2(1Δg)

triplet states 3D-TPP 3D-PdTPP 2D-TPP TPP

τT0 (μs)

kO2 (M−1 s−1)

f air T

ΦΔ

τΔ/μs

490 200 460 68

× × × ×

>0.999 0.998 >0.999 0.997

0.58 0.56 0.67 0.60b

75 78 77 75

1.4 1.3 1.9 1.6

9

10 109 109 109

τT0 stands for the lifetime of the porphyrin triplet states in argonsaturated acetonitrile; kO2 is the bimolecular rate constant of the triplet state quenching by oxygen; f air T = 1 − τT/τT0, i.e., it is the fraction of the triplet states quenched by oxygen in air-saturated acetonitrile; ΦΔ is the quantum yield of singlet oxygen formation; τΔ is the lifetime of O2(1Δg). bLiterature value.33 a

oxygen, the fluorescence band at 609 nm is accompanied by the phosphorescence bands at 699 and approximately 765 nm (Figure 2). The phosphorescence lifetime, corresponding to the lifetime of the triplet states, is 200 μs in an oxygen-free environment.

Figure 2. Phosphorescence emission spectra of 3D-PdTPP dispersions in acetonitrile saturated by air (a) and in the absence of oxygen (b). The excitation wavelength was 410 nm. Inset: Corresponding luminescence band of O2(1Δg) upon 520 nm excitation. Figure 1. Normalized absorption spectra of 2D-TPP (a), 3D-TPP (b), and 3D-PdTPP (c) dispersed in acetonitrile compared with the spectrum of TPP in acetonitrile (d).

The formation of the triplet states of 2D-TPP and 3D-TPP was monitored by transient absorption spectroscopy at 460 nm (SI Figure S11). Interestingly, the lifetimes of the triplet states (τT0) for metal-free COFs are several times longer than that of molecular TPP, indicating the reduction of nonradiative relaxation processes due to the limited contact of the incorporated porphyrin units with the solvent (Table 2). The results document that the TPP and PdTPP units, constituting the COF backbone, produce the long-lived triplet states after excitation by visible light. Transient absorption spectra of these triplet states have a broad maximum around 460 nm, resembling the spectrum of molecular TPP in solution (SI Figure S11D). The triplet states of COFs are considerably quenched by molecular oxygen as documented by disappearance of

the sharp Soret bands of monomeric TPP at 413 nm, the porphyrin units constituting the polymer backbone are broadened and red-shifted, indicating interactions between the porphyrin units in these materials. Similarly, the Q bands exhibit a red-shift. The Q(0,0) and Q(0,1) fluorescence bands are broadened and slightly red-shifted when compared to the corresponding bands of TPP (SI Figure S10). Also, fluorescence lifetimes are shortened, and in contrast to TPP with a lifetime 9.20 ns, the fluorescence decay curves are biphasic with two components: 1.94 (9%) and 11.9 (91%) ns for 3D-TPP and ≤0.4 (10%) and 9.02 (90%) ns for 2D-TPP. Because Pd-porphyrin moieties are 8531


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diphenylanthracene was 79, 85, and 87% in the first, second, and third run, respectively (Figure S13). Thus, the O2(1Δg) productivity was preserved within the experimental error, documenting the photochemical stability of 3D-PdTPP similarly to the stability reported previously for covalently bound porphyrin networks connected via C−C bonds.12 The presented results prove the formation of O2(1Δg), its oxidative potential, and material photostability therefore unambiguously indicate that investigated porphyrin-based COFs can be considered as promising O2(1Δg) photosensitizers. 3.4. Photodynamic Inactivation of Bacteria. We employed two conditional pathogens with a well-known ability to form biofilms for testing the antibacterial efficacy of the coatings made of COFs. Pseudomonas aeruginosa is G− aerobic rod bacterium ubiquitously present in nature. The growth of P. aeruginosa biofilms on hospital equipment, including catheters and ventilators, could seriously endanger patients with burns, immunocompromised patients, or newborns.37 Enterococcus faecalis is G+ round facultative anaerobic bacterium often forming chains. It is a natural component of human colonic microflora and a dangerous conditional pathogen due to its unusually high natural resistance to antibiotics and environmental stresses.38 It is a source of nosocomial infections of urinary and bile tract. Both bacterial strains formed single-cell-thick stable biofilms after 24 h and multicell-thick biofilms after 48 h of incubation at room temperature. The growth of bacteria on the COF coatings occurred mostly in the gaps between COF domains, thus seemingly decreasing the relative density of bacteria even in the dark, compared to the blank coatings without COFs (Figure 4A,B and SI Figure S14). This effect is pronounced in case of P. aeruginosa with bigger cells and forming less dense biofilms. In contrast to the biofilm growth in the dark, the formation of biofilms was completely inhibited under irradiation with either a 460 nm LED light source (Figure 4A,B) or halogen lamp (SI Figure S15). On the basis of the above-described photophysical properties of COFs, these effects can be attributed to their O2(1Δg) photosensitizing activity. The antibacterial function induced by 525 nm light toward P. aeruginosa was limited to the 3D-TPP and 3D-PdTPP coatings (Figure 4A), probably due to lower intensity of 525 nm light (7 mW cm−2) compared to 460 nm light (20 mW cm−2). The photostability of the COF coatings was assessed by measuring the antibacterial effects after 48 h of continuous irradiation with 460 nm light (Figure 4C). Clearly, all the coatings remain antibacterial, however, only the 3D-TPP coatings maintained the same magnitude of the antibacterial effect, indicating their superior long-lasting activity. In addition, we excluded the release of porphyrins from the material during the bacterial photoinactivation experiments. It implies that the imino bonds within the COFs structure are stable when exposed to aqueous environment and irradiation, therefore, the antibacterial effect is caused by the photosensitizing activity of COFs and not by their partial dissolution. All three COFs are also effective in destroying already existing biofilms and killing biofilm cells. As documented in Figure 4D, the biofilms formed during 24 h incubation in the dark were completely destroyed after 4 h irradiation with 460 nm light. Because the experiments investigating inhibition of the biofilm formation were performed at relatively high total energies, the efficacy of the most effective COF, 3D-TPP, was

phosphorescence bands of 3D-PdTPP (Figure 2), high rate constants kO2, and high fractions of the porphyrin triplet states trapped by oxygen f air T (SI Figure S11, Table 2). These results initiated the investigation of the production of O2(1Δg) by measuring its characteristic luminescence in the near-infrared region. As anticipated, all three COFs exhibit a luminescence band of O2(1Δg) at approximately 1270 nm (Figure 2, inset), confirming the formation of O2(1Δg) in acetonitrile dispersions upon excitation at the Soret or Q bands of the embedded porphyrin units. The temporal profiles of O2(1Δg) luminescence allowed the evaluation of O2(1Δg) lifetimes, τΔ, after fitting to a single-exponential function with the exclusion of the initial part, governed by the O2(1Δg) formation kinetics (Table 2, SI Figure S12). The obtained O2(1Δg) lifetimes correspond with the reported O2(1Δg) lifetime in acetonitrile,36 indicating a minimal, if any, self-quenching effect of COFs toward O2(1Δg). Comparison of O2(1Δg) luminescence amplitudes of the COF dispersions with that of TPP solution allowed estimation of the ΦΔ values, even though the increased light scattering level of COF dispersions causes some uncertainty (Table 2). The analysis indicates that COF materials remain good photosensitizers and their production of O2(1Δg) is fully comparable with that of individual TPP molecules in organic solvents. The measured photosensitizing activity of COFs is surprising because high local concentrations of individual porphyrin molecules, e.g., in aggregates, leads to considerable diminishing of the O2(1Δg) production due to fast competitive relaxations of the excited states. Evidently, the arranged porphyrin units in COFs appear to minimally affect their photosensitizing activity, indicating that COFs may produce high local concentrations of O2(1Δg) needed for inactivation of bacteria. 3.3. Photosenzitizing Properties. The ability of O2(1Δg), produced by COFs, to oxidize external targets was evaluated by the photooxidation reaction of 9,10-diphenylanthracene to the corresponding endoperoxide. As shown in Figure 3, 3D-PdTPP

Figure 3. Reaction of photosensitized O2(1Δg) produced by COF dispersions with 9,10-diphenylanthracene in acetonitrile. Blank experiments performed in the absence of COFs excluded any photoreaction of 9,10-diphenylanthracene itself.

is the most efficient photooxidative material. The efficiency evidently originates from the composition of COFs rather than from the textural properties because 3D-PdTPP and 3D-TPP with surface areas of tens m2 g−1 photooxidize the substrate more effectively than two-dimensional 2D-TPP with a surface area of 475 m2 g−1. To prove the photostability of COFs, 3DPdTPP was separated from the solvent, washed, and reused twice. After 3 h of irradiation, the conversion of 9,108532


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Figure 4. Antimicrobial function of the COF coatings. The surfaces were incubated 24 h in the dark (black) and under 460 (20 mW cm−2, blue) or 525 nm irradiation (7 mW cm−2, green) with P. aeruginosa (A) and E. faecalis (B). A similar experiment was performed after 48 h incubation under 460 nm light (C). To test the direct killing of cells in the biofilms, the biofilms of P. aeruginosa and E. faecalis were established by 24 h incubation in the dark, followed by 4 h irradiation with 460 nm light (D). The blank experiments were performed with the polymer coating without COFs. In all cases, the amount of the biofilm is quantified as percentage of the surface covered with bacteria (y axis). The experiments were analyzed by the Student t test, and the results with p < 0.01 (labeled as *) are considered as significant.

inate over the formation of the excited states responsible for the generation of O2(1Δg). The design of the antibacterial coatings made of COFs is promising because COFs are photostable and have high quantum yields of O2(1Δg), allowing their effective application for a long period.

evaluated using considerably lower light intensity of 1 mW cm−2 (SI Figure S16), i.e., 20 times lower intensity than that used in the experiment described in Figure 4A. Also in this case, the formation of the P. aeruginosa biofilm was fully inhibited, whereas no effect was observed on destruction of the already established biofilm. We found strong photoantibacterial effects of COFs, especially of 3D-TPP, which exhibited high photostability, broad spectral efficiency, and good dispersibility in the polystyrene coating. Importantly, the effect of COFs deposited on the surface was only local as we never observed any change in the cell density of the bacterial suspension in the dish with immersed glass plates nor any inhibitory effect on the formation of the biofilm on the other side of the plate. The presence of basic amino groups in all materials, as indicated by FTIR, appears to be advantageous for the PDI application because in this way the surface of the photosensitizers can interact strongly with the bacteria.39

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b19835. Syntheses of precursors, solid state characterizations, fluorescence, transient spectroscopy, singlet oxygen, biocidal effects (PDF)

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AUTHOR INFORMATION

Corresponding Author

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*E-mail: [email protected].

CONCLUSIONS In conclusion, we successfully synthesized and characterized porphyrinic COFs bound together by the Schiff-base reaction with the 2D and 3D topology. The COFs produce O2(1Δg) in yields fully comparable with those of TPP molecules in organic solvents, suggesting that the photosensitizing activities of the COFs are not affected by high local concentrations of individual porphyrin units in the structure. On the basis of this finding, the COFs are materials producing high localized O2(1Δg) fluxes, needed for inactivation of bacteria on surfaces. This propensity is advantageous when compared with the behavior of molecular porphyrin sensitizers. In such cases, the production of O2(1Δg) is strongly reduced by the formation of aggregates, especially at higher porphyrin concentrations, in which nonradiative relaxation channels are fast and predom-

ORCID

Pavel Kubát: 0000-0002-7861-9212 Jan Demel: 0000-0001-7796-6338 Kamil Lang: 0000-0002-4151-8805 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Czech Science Foundation (no. 16-15020S), the Operative program PragueCompetitiveness (OPPC CZ.2.16/3.1.00/21537, OPPC CZ.2.16/ 3.1.00/24503), and the National Program of Sustainability (NPU I LO1601). J.H. gratefully acknowledges the Charles University Grant Agency (no. 252216) for financial support. 8533


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