Synthesis of Self-Assembled Porphyrin Nanoparticle Photosensitizers

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Synthesis of Self-Assembled Porphyrin Nanoparticle Photosensitizers Dong Wang, Lijuan Niu, Zeng-Ying Qiao, Dong-Bing Cheng, Jiefei Wang, Yong Zhong, Feng Bai, Hao Wang, and Hongyou Fan ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b01010 • Publication Date (Web): 03 Apr 2018 Downloaded from http://pubs.acs.org on April 3, 2018

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ACS Nano

Synthesis

of

Self-Assembled

Porphyrin

Nanoparticle Photosensitizers Dong Wang1,2, Lijuan Niu1,2, Zeng-Ying Qiao3, Dong-Bing Cheng3, Jiefei Wang1,2, Yong Zhong1,2, Feng Bai,1,2,* Hao Wang3,* and Hongyou Fan4, 5,*

1

Key Laboratory for Special Functional Materials of the Ministry of Education, Henan University, Kaifeng 475004, P. R. China;

2

Collaborative Innovation Center of Nano Functional Materials and Applications, Henan University, Kaifeng 475004, China;

3

CAS Key Laboratory for Biological Effects of Nanomaterials and Nanosafety, National Center for Nanoscience and Technology (NCNST), Beijing 100190, China; 4

Department of Chemical and Biological Engineering, the University of New Mexico, Albuquerque, New Mexico 87106, United States; 5

Sandia National Laboratories, Albuquerque, New Mexico 87185, United States.

Corresponding Author F.B. ([email protected]), H.W ([email protected]), H.F. ([email protected])

ACS Paragon Plus Environment

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Abstract

The use of nanoparticles as a potential building block for photosensitizers has recently become a focus of interest in the field of photocatalysis and photodynamic therapy. Porphyrins and their derivatives are effective photosensitizers due to extended π-conjugated electronic structure, high molar absorption from visible to near infrared spectrum, and high singlet oxygen quantum yields as well as chemical versatility. In this paper, we report a synthesis of selfassembled porphyrin nanoparticle photosensitizers using zinc meso-tetra (4-pyridyl) porphyrin (ZnTPyP) through a confined noncovalent self-assembly process. Scanning electron microscopy (SEM) reveals formation of monodisperse cubic nanoparticles. UV-vis characterizations reveal that optical absorption of the nanoparticles exhibit red shift due to non-covalent self-assembly of porphyrins, which not only effectively increase intensity of light absorption but also extend light absorption broadly covering visible light for enhanced photodynamic therapy. Electron spinresonance spectroscopy (ESR) studies show the resultant porphyrin nanoparticles release high yield of singlet oxygen. Nitric oxide (NO) coordinates to central metal Zn ions to form stabilized ZnTPyP@NO nanoparticles. We show that under light irradiation, ZnTPyP@NO nanoparticles release highly reactive peroxynitrite molecules that exhibit enhanced antibacterial photodynamic therapy (APDT) activity. The ease of the synthesis of self-assembled porphyrin nanoparticles and light triggered release of highly reactive moieties represent a completely different photosensitizer system for APDT application.

Keywords: self-assembly, porphyrin nanoparticles, antibacterial photodynamic therapy, APDT, photosensitizers


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The use of photoactive nanoparticles as a potential building block for photocatalysts and photosensitizers (PS) has recently become a focus of interest in the field of photodynamic therapy (PDT) that is widely studied and established for the treatment of malignant tissues such as cancer.1-11 PDT is based on the use of nontoxic dye compounds called PS that release reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radicals (•OH), superoxide radicals (O2•), or singlet oxygen (1O2) upon selective irradiation of the photosensitizers by a specific wavelength of light.12 These reactive species are the key factors in PDT to destroy cancer cells and microorganisms. Effectiveness of PDT essentially depends on several characters of PS including high absorption coefficient, high photo stability, and no toxicity under no illumination. Among previously studied PS, Metal nanoparticles, such as gold and silver nanoparticles have been demonstrated for PDT based on their surface plasmonic behavior.7-11 However, extra steps of surface functionalization are required to ensure biocompatibility.10 Additionally, high cost limits their large-scale practical applications. Porphyrins and phthalocyanines have been also investigated as an important part of PDT producing ROS upon exposure to light in the presence of oxygen. Porphyrins and their derivatives have excellent photo physical properties as outstanding PS because of their very intense absorption bands in the visible region and high singlet oxygen quantum yield due to their large π-conjugated aromatic domains.13-17 They are widely distributed in organism and possess low toxicity under no illumination, which implies their good biocompatibility. Despite of previous efforts, ability to produce porphyrin nanoparticle PS is more desirable to improve PDT because nanosize provides high surface area for cell interactions and faster transport. In addition, creation of other reactive species such as nitric oxide led to significantly enhanced biocidal activity. NO is a naturally occurring bioactive molecule with well-established physiological functions in the cardiovascular


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and immune systems.18-21 It is a signaling molecule that induces inflammation in low doses.22 As the concentration increases, NO is capable of destroying or inhibiting bacteria by forming compounds that react with DNA and inhibit its repair.23, 24 Additionally, NO has been noted to both enhance and modulate the effects of other chemotherapies.25-27 Therefore, it will be highly desirable to fabricate biocompatible nanoscale particle photosensitizers that are able to produce both NO and ROS for more effective PDT. Herein we report a synthesis of porphyrin-nitric oxide nanoparticle system that can produce ROS and release NO simultaneously under light irradiation. The porphyrin-nitric oxide nanoparticles exhibit significantly enhanced antibacterial activity due to the release of NO and ROS. Porphyrin nanoparticles were synthesized using ZnTPyP through a confined noncovalent self-assembly process.28,

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Subsequently, NO coordinates to central Zn ions to form

ZnTPyP@NO nanoparticles. Using light as the trigger, the antibacterial abilities of ZnTPyP and ZnTPyP@NO nanoparticles have been studied. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were used as the pathogenic bacteria to demonstrate the antibacterial efficiency. Under light irradiation, the ZnTPyP@NO nanoparticles produced ROS and release NO at the same time, leading to significantly enhanced biocidal activity while ZnTPyP nanoparticles only produce ROS. Neither ZnTPyP nanoparticles nor ZnTPyP@NO nanoparticles shown antibacterial activity in dark. We found that the ZnTPyP@NO nanoparticles are chemically stable and they shown no loss of NO after stored in the dark at room temperature for 180 days.

Results and Discussion


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We chose ZnTPyP as the building block to synthesize self-assembled nanoparticle PS because its absorption bands are situated in the visible range from 400 to 650 nm so that visible light can be used to initiate photoreactions of ZnTPyP nanoparticles to release ROS. Our previous work showed that NO can coordinate to central Zn ions of ZnTPyP and form [email protected] We showed that ZnTPyP@NO nanoparticles are very stable in dark at room temperature. In this nanoparticle system, ZnTPyP is not only the PS, but also the carrier of NO. Under sunlight irradiation, ZnTPyP@NO nanoparticles transfer the light energy to surrounding oxygen to produce ROS and also trigger the release NO. NO in combination with O2•- leads to highly reactive peroxynitrite molecules enhances significantly the antibacterial activity (Scheme 1). In a typical preparation, 9.1 mL of aqueous solution containing cetyltrimethyl ammonium bromide (CTAB) (0.011M) and NaOH (0.0027 M) was prepared at room temperature. Then we injected 0.45 mL fresh stock ZnTPyP solution (0.01 M ZnTPyP dissolved in 0.05 M HCl solution) into it and stirred for 48 hrs. The final nanoparticles were collected by centrifugation at 12000 rpm. As illustrated in Figure 1, the resultant nanoparticles have well-defined cubic morphologies. Figure 1A shows a representative scanning electron microscopy (SEM) image of the ZnTPyP nanoparticles that are monodisperse with narrow size distribution. The average size of the nanoparticles is approximately 40 nm. The self-assembly process was driven by noncovalent interactions such as hydrophobic-hydrophobic interactions, π-π stacking between molecules or surfactants,28,

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which initiates nucleation and growth of J-aggregate ZnTPyP

nanostructures. Transmission electron microscopy (TEM) reveals a uniform morphology in these J-aggregate nanostructures without defects (Figure 1B). These nanoparticles are very well dispersed in water to form a clear and transparent solution (Figure


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Scheme 1. Schematic illustration of synthesis of the self-assembled ZnTPyP nanoparticles, ZnTPyP@NO nanoparticles, and the APDT activity of ZnTPyP@NO nanoparticles under light.

1C). The ZnTPyP nanoparticles exhibit optical properties that are quite different from the ZnTPyP monomers. Figure 1D shows the absorption spectra of the ZnTPyP nanoparticles and monomers. The intense Soret band of the ZnTPyP monomers (at ~ 424 nm) become split after self-assembly with a red-shifted band arising (at ~ 442 nm), indicative of J-aggregation. The absorption bands of nanoparticles are located in the visible range between 400 and 650 nm. The absorption spectra of the ZnTPyP nanoparticles cover a broader visible region of the spectrum than that of ZnTPyP monomers. For B-band, ZnTPyP monomers range from


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Figure 1. Characterizations of the self-assembled ZnTPyP nanoparticles. (A) SEM of the ZnTPyP nanoparticles that were prepared using 0.45 mM ZnTpyP and 10 mM CTAB at pH around 8 stirring for 48 hrs. (B) Corresponding TEM image of the ZnTPyP nanoparticles. (C) Optical picture of aqueous dispersion of the ZnTPyP nanoparticles. (D) UV-vis absorption of ZnTPyP monomers dissolved in DMF (black) and assembled ZnTPyP nanoparticles dispersed in water (red).

390 to 444 nm. However, ZnTPyP nanoparticles range from 375 to 545 nm, which is 3 times broader than ZnTPyP monomers. It can be deduced that ZnTPyP nanoparticles can employ visible light more effectively.


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We have discovered that ZnTPyP nanoparticles show a specific chemical sorption of NO.28 NO was absorbed into self-assembled ZnTPyP nanoparticles to form the ZnTPyP@NO (see details in Experimental Section). The Fourier transform infrared (FTIR) spectroscopy of the ZnTPyP@NO nanoparticles demonstrated that NO coordinates to central Zn ions, which was evidenced by the typical peaks of Zn-NO band stretching vibration located at 1382.5 cm-1 (Figure 2A(b)). In order to further confirm the NO absorption, thermogravimetric analysis (TGA) on the specimens after exposure to NO was performed. The ZnTPyP@NO and the ZnTPyP nanoparticles were heated at speed of 10 ˚C/min from 30 ˚C to 210 ˚C. After that, the nanoparticles were kept at 210 ˚C for 60 mins. The ZnTPyP@NO nanoparticles show a consistent weight loss from 110 ˚C to 190 ˚C, however the weight of ZnTPyP nanoparticles remains constant (see Figure 2B). As shown in Figure 2B, the ZnTPyP@NO nanoparticles lose 20 wt% compared with the ZnTPyP nanoparticles after keeping at 210 ˚C for 60 mins. FTIR spectroscopy results suggest that the typical peaks of Zn-NO band stretching vibration located in 1382.5 cm-1 almost disappeared after heated to 210 ˚C and ZnTPyP nanoparticles are stable without chemical degradation and ZnTPyP@NO nanoparticles release NO only between 110 and 210 ˚C (Figure 2A(c)). It can be concluded from the TGA results that the ZnTPyP nanoparticles show

20 wt% NO absorption. The UV-vis absorption bands of the ZnTPyP nanostructures

change slightly after absorbing NO (Figure 2C). After NO absorption, the absorption spectra of the nanoparticles are still located in the visible range between 400 and 650 nm, just the B bands split to 410 nm and 460 nm two peaks. The Q bands red shift in comparison with ZnTPyP nanoparticles. Previous studies have shown that some materials such as silica nanoparticles modified with NO donors are effective in NO absorption and release.30, 31 However, these materials are required


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to be stored in a harsh environment, like vacuum-sealed, dark container at - 20 ˚C to prevent from random release of NO, which limits their use.32 It is important for PS to be stable at room temperature for effective APDT. We found that the ZnTPyP@NO nanoparticles are very stable


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Figure 2. Characterizations of NO absorption by ZnTPyP nanoparticles. (A) FTIR spectra of (a) ZnTPyP and (b) ZnTPyP@NO nanoparticles before TGA measurements and (c) ZnTPyP@NO, (d) ZnTPyP after TGA measurements. (B) TGA results for ZnTPyP (black) and ZnTPyP@NO (red) nanoparticles (The temperature increased from 30 to 210 ˚C at speed of 10 ˚C/min and kept at 210 ˚C for 60 mins). (C) UV-vis absorption of ZnTPyP (black) and ZnTPyP@NO (red) nanoparticles dispersed in water.

in dark at ambient conditions. After the nanoparticles were stored in dark for six months at ambient conditions, the UV-vis spectra of the nanoparticles (Figure S1) showed almost no changes. To verify whether the NO are still coordinated to the central Zn ions, FTIR spectroscopy was also performed on these nanoparticles and shown that the typical peaks of ZnNO stretching vibration of ZnTPyP@NO nanoparticles located at 1382.5 cm-1 still existed (Figure S2), which indicates that NO still coordinates to the central Zn ions in ZnTPyP@NO nanoparticles with high stability, favorable their storage, transportation, and use. The generation of singlet oxygen from the self-assembled ZnTPyP nanoparticles was measured by ESR. ZnTPyP nanoparticles were dispersed in water to explore the singlet oxygen generation and 2,2,6,6-tetramethyl-4-piperidone (TEMPO) was employed as the spin-trapping reagent. As shown in Figure 3A, under the white light, a characteristic 1:1:1 triplet ESR signal was detected, which is typically attributed to the ESR spectrum of singlet oxygen.33 This suggests that singlet oxygen could be generated by the ZnTPyP nanoparticles effectively. Although the absorption bands of the ZnTPyP nanostructures change a little after absorbing NO, they are still located mainly in the visible range between 400 and 650 nm.


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Figure 3. (A) ESR spectra of 4-oxo-TEMP adduct with singlet oxygen generated in the H2O (black) and ZnTPyP nanoparticles (red) under the white light for 90 s. (B) FTIR spectra of ZnTPyP@NO in dark (black) and illuminated 2 hrs with visible light (red). (C) UV-vis absorption of the ZnTPyP@NO nanoparticles under light illumination for different time. (D) Fluorescence of L-tyr in the system before (black) and after (red) adding ZnTPyP@NO (λex = 313 nm), and illuminated with visible light for two minutes (blue).

Most interestingly, we found that visible light can set the NO free effectively. FTIR spectroscopy suggested that the typical peaks of Zn-NO band stretching vibration of ZnTPyP@NO located in 1382.5 cm-1 almost disappeared after illumination with visible light for only 2 hrs (Figure 3B),


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indicating that NO has been released completely within the time scale. To explore the process of NO release from the ZnTPyP@NO, the change of the UV-vis absorption of the ZnTPyP@NO particles under the visible light has been researched (Figure 3C). With the increase of illumination time, the UV-vis absorption reduced gradually, which implied that NO was released gradually. For further study, we used the Griess assay protocol to explore the NO release.34 NO deactivation in aqueous solutions generates NO2-, which can react with the Griess reagent and produce a kind of coloration for detection. The resulting coloration is quantified using UV-vis spectroscopy, and the characteristic absorption band of Griess agent at 520 nm increased when illumination time increase, which can be concluded that NO is released gradually with the increase of illumination time (see Figure S3). Under light exposure, ROS and NO show synergistic effect.35 The ZnTPyP@NO nanoparticles may generate a kind of more effective species: peroxynitrite ion (ONOO-).36 ONOO- can be generated from the reaction of NO released from the ZnTPyP@NO and superoxide O2•- produced by ZnTPyP@NO under light irradiation. Its good stability in aqueous solutions (half-life time 10 - 20 ms) is a great advantage so that it can destroy cellular constituent effectively and induce cell death.37 Presence of ONOO- was verified by using a fluorescence method.38 L-tyrosine was used as a probe molecule in the presence of CO2 in weak alkaline environment. Dimerization of tyrosine (Dityr) will be generated by oxidation of ONOO-.39, 40 The excitation wavelength (λex) of Dityr is 313 nm and the emission wavelength (λem) of Dityr is 406 nm. In a typical experiment, a 10 mL of aqueous solution containing PBS (0.10 M, pH = 8.2), NaHCO3 (0.015 M) and L-tyrosine (5. 0× 10-4 M) was prepared at room temperature. Then 1 mg ZnTPyP@NO nanoparticles were added into the solution, and illuminated with visible light for two minutes. As shown in Figure 3D, L-tyr in the system before and after adding ZnTPyP@NO particles, shown no fluorescence signal. However,


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the fluorescence intensity at 406 nm of Dityr in the system with presence of ZnTPyP@NO was very strong, after illuminated for 2 mins by visible light. This suggested that visible light can trigger generation of ONOO- from ZnTPyP@NO nanoparticles, which is evidenced by the dimerization of tyrosine. The antibacterial activity of the nanoparticles was studied under light irradiation. A solarsimulator was used to mimic natural sunlight to explore the efficiency of antibacterial activity and the suspended cells of E. coli and S. aureus in nutrient medium were used as models. We found that both ZnTPyP@NO nanoparticles and ZnTPyP nanoparticles dispersed in water can generate a packed film on the glass. We dipped the film glasses into the bacterial cells suspension (OD600nm = 0.1) and incubated them for 1 hr under the simulated sunlight to kill the bacteria. After that, the film glasses were imprinted on TSB solid medium to incubate for 24 hrs in the dark at room temperature. The ZnTPyP nanoparticles film glass and the control film glass under the same condition were also explored. It can be concluded that the ZnTPyP@NO nanoparticles can kill the bacterial cells effectively (see Figure S4). To further study the antibacterial efficiency of the nanoparticles, 100 µg ZnTPyP nanoparticles or ZnTPyP@NO was added into nutrient medium (1 mL) that contain bacterial cells (C = 105 CFU/mL), followed by putting 200 µL of it into 96-well plates. We exposed the suspensions to the simulated sunlight at different intervals (0, 15, 30, 60, and 120 mins), and then the suspensions were put into shaking tables for 6 hrs at 37 ˚C. After that, the bacterial viability was determined by OD600 nm using multifunctional microplate reader. As shown in Figure 4, ZnTPyP nanoparticles and ZnTPyP@NO nanoparticles did not exhibit antibacterial property in the dark. However, under light irradiation, these nanoparticles show antibacterial effect. With increasing irradiation time, the antibacterial effect of ZnTPyP nanoparticles and ZnTPyP@NO nanoparticles were enhanced


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notably. However, although the ZnTPyP nanoparticles can also kill bacteria, the ZnTPyP@NO nanoparticles exhibited more significant antibacterial capacity. Especially for the S. aureus, with only 30 mins light illumination, ZnTPyP@NO nanoparticles killed almost all of the S. aureus. In contrast, ZnTPyP nanoparticles killed only half of the S. aureus. It took 120 mins for the

Figure 4. Characterizations of antibacterial activity under visible light. Time dependence of biocidal action against suspended cells of S. aureus (A) and E. coli (B) for treatments with simulated sunlight alone as reference (black columns), and for treatments with simulated sunlight in the presence of suspended nanoparticles of ZnTPyP (red columns), ZnTPyP@NO (blue columns). Error bars represent standard deviation (n =3).

ZnTPyP nanoparticles to kill all the S. aureus, which is four times longer than ZnTPyP@NO nanoparticles. These results unambiguously establish that the ZnTPyP@NO nanoparticles are more powerful PS to kill bacteria than ZnTPyP nanoparticles, which is attributed to the simultaneous release of NO and ROS from ZnTPyP@NO nanoparticles while ZnTPyP particles only release ROS.


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In comparison with TiO2 that is widely studied as an antibacterial agent under light irradiation, ZnTPyP@NO took less illumination time and less mass concentration to kill S. aureus more effectively.41 For the case of E. coli, about 50% of bacteria was killed by ZnTPyP@NO nanoparticles with 30 mins illumination, and all of the E. coli was killed with 120 mins illumination. In contrast with TiO2, under the same light conditions, only 82% of the E. coli was killed with 120 mins illumination. Additionally, TiO2 has dark toxicity and can’t be controlled without light.42 Compared with composite mesoporous silica materials that produce both NO and ROS,35 under the same light conditions (120 mins illumination), less amounts of ZnTPyP@NO nanoparticles performed better antibacterial activity.

Conclusions In summary, we synthesized self-assembled porphyrin nanoparticle photosensitizers using ZnTPyP through a confined noncovalent self-assembly process. Through sorption of NO, ZnTPyP@NO nanoparticles become not only a PS, but also a NO carrier. Upon light illumination, both ZnTPyP nanoparticles and ZnTPyP@NO nanoparticles killed bacteria effectively. With release of NO together with ROS, ZnTPyP@NO nanoparticles are more powerful than ZnTPyP nanoparticles. One important feature for the ZnTPyP@NO nanoparticles is that they are fairly stable for long time in dark at ambient conditions, which is advantageous for storage and transportation. In addition, self-assembly enables the formation of J-aggregates to extend optical spectra covering broader visible light, which is also critical for effective APDT.

Experimental Section


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Materials. Zinc meso-tetra(4-pyridyl) porphyrin (ZnTPyP) was purchased from Frontier Scientific, Inc. Cetyltrimethyl Ammonium Bromide (CTAB) was purchased from Aldrich Chemical Co. and used without further purification. Hydrochloric acid (HCl 1M) and Sodium hydroxide (NaOH 1M) solution were purchased from Aldrich Chemical Co. All the solutions were prepared in ultrapure water (resistivity of 18.2 MΩ·cm). NO was purchased from Zhengyuan Co. TSB was purchased from Aoboxing Bio-TECH co. The solar-simulator was purchased from Perfectlight Co. L-tyrosine was purchased from Lablead Co. Bacterial strains of S. aureus (ATCC 6538), E. coli (ATCC 8739), were obtained from China General Microbiological Culture Collection Center.

Materials preparation. The ZnTPyP nanoparticles were synthesized through a simple solution procedure. In a typical preparation, 0.45 mL fresh stock ZnTPyP solution (0.01 M ZnTPyP dissolved in 0.05 M HCl solution, and the mixture was stirred for 30 min before using) was quickly added into 9.1 mL of continuously stirred aqueous solution of CTAB (0.011M) and NaOH (0.0027 M) at room temperature (25 ˚C). Then the mixture was stirred for 24 hrs. The green solution was centrifuged at 12000 rpm and washed twice with Millipore water to remove the surfactants. The ZnTPyP@NO nanoparticles were synthesized through a procedure as follows: The ZnTPyP nanoparticles were freeze-dried and put into a reaction kettle, pumped vacuum, then we put NO gas into it at 10 atm and kept it for 7 days to ensure that NO effectively coordinates to central Zn ions of ZnTPyP.


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Characterization. The powders were re-dispersed in pure water and deposited on silicon wafer substrates and TEM grids for the characterization by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM images were taken using a Hitachi 5200 FEG microscope. TEM was performed on a JEOL 2010 with 200 kV acceleration voltage, equipped with a Gatan slow scan CCD camera. The UV-vis measurements were performed using a Cary 50 Conc UV-vis. TGA was performed on a PerkinElmer STA 800 Simultaneous Thermal Analyzer. FTIR was performed on a PerkinElmer Frontier FT-IR/FIR Spectrometer. 1

O2 measured by ESR. The generation of 1O2 from the self-assembled ZnTPyP nanoparticles

was measured by ESR. Under the white light for 90 s, ZnTPyP nanoparticles (70 µg·mL-1) were dispersed in water to explore the 1O2 generation and 2,2,6,6-tetramethyl-4-piperidone (TEMPO) was employed as the spin-trapping reagent to detect a characteristic 1:1:1 triplet ESR signal. NO detection by Griess reagent. To characterize the NO released from the ZnTPyP@NO nanoparticles during light exposure, a modified Griess assay was performed.34 ZnTPyP@NO nanoparticles was dissolved in 1 M NaOH (pH = 14) for detection of NO release. ONOO- detection by fluorescence method. L-tyrosine was used as a probe molecule in presence of CO2 in weak alkaline environment. Dimerization of tyrosine (Dityr) will be generated by oxidation of ONOO-. In a typical experiment, a 10 mL of aqueous solution containing PBS (0.10 M, pH = 8.2), NaHCO3 (0.015 M) and L-tyrosine (5.0 × 10-4 M) was prepared at room temperature. Then 1 mg ZnTPyP@NO nanoparticles were added into the solution, and illuminated with visible light for two mins. Bacteria viability assay. E. coli and S. aureus were cultured respectively in the TSB medium at 37 ˚C on a shaker bed at 200 rpm for 4 − 6 hrs. Then the concentration of bacteria, corresponding


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to an optical density of 0.1 at 600 nm for 1 × 108 CFU mL−1 diluted with the medium, was measured by UV−vis spectroscopy (Cary100Bio). We added the bacterial suspension (20 µL of 1 × 106 CFU mL−1) to medium (180 µL of 111 µg mL−1 ZnTPyP, 111 µg mL−1 ZnTPyP@NO) respectively for each well. Then PBS (200 µL) was added as the blank control and the 96-well plates were shaken at 37 ˚C on a shaker bed with 200 rpm for 18 hrs. The bacterial viability was determined by OD600 nm using multifunctional microplate reader (Tecan infinite M200). Each concentration was measured in triplicate, and all experiments were repeated at least thrice in parallel.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Detailed characterization including FTIR and UV− vis, and APDT results.

AUTHOR INFORMATION Corresponding Author Corresponding author emails, phone numbers, and fax numbers: F.B. ([email protected]), Tel: 86-15039024866, Fax: 86-0371-23883868 H.W ([email protected]), Tel: 86-18611786240, Fax: 86-010-62656765 H.F. ([email protected]), Tel: (505) 272-7128, Fax: (505) 272-7336

Author Contributions


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H.F. conceived the idea. D.W., L.N, Z.W., D.C., J.W., Y.Z., H.W. and F.B. performed the experiments and collected/analyzed data. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare no competing financial interest.

ACKNOWLEDGMENTs This work was supported by the U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering. F.B. acknowledges the support from the National Natural Science Foundation of China (21422102, 21771055, U1604139, 21171049), Plan for Scientific Innovation Talent of Henan Province (No. 174200510019), and Program for Changjiang Scholars and Innovative Research Team in University (No.PCS IRT_15R18). Sandia National Laboratories is a multimission laboratory managed and operated by National Technology and Engineering Solutions of Sandia, LLC., a wholly owned subsidiary of Honeywell International, Inc., for the U.S. Department of Energy’s National Nuclear Security Administration under contract DE-NA0003525.

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