MetalâOrganic Framework@Porous Organic Polymer Nanocomposite

Article pubs.acs.org/cm

Metal−Organic Framework@Porous Organic Polymer Nanocomposite for Photodynamic Therapy Xiaohua Zheng,†,‡ Lei Wang,*,† Qing Pei,†,‡ Shasha He,†,‡ Shi Liu,† and Zhigang Xie*,† †

State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, P. R. China ‡ University of Chinese Academy of Sciences, Beijing 100049, P. R. China S Supporting Information *

ABSTRACT: The limitation for the biomedical application of porous organic polymers (POPs) is the big size and poor dispersibility in aqueous media. Herein, a nanoscale metal−organic framework (MOF)@POP composite, named UNM, has been synthesized by epitaxial growth of the photoactive porphyrin-POPs (H2P-POP) on the outer surface of amine containing UiO-66 (UiO-AM). After the growth of POPs, the crystallization, pore structure, and size distribution of UNM are retained well. The formed UNM possesses a small size of less than 200 nm and could be internalized by cancer cells. Such light-activated UNM exhibits efficient ability to generate 1O2 under various experimental conditions, which can be further applied for PDT efficacy. The present work demonstrates the great potential of nanoscale porous polymers in biomedical fields and cancer treatment.

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INTRODUCTION Photodynamic therapy (PDT) has gained wide scientific attention due to its minimally invasive nature and tiny collateral damage to surrounding normal tissues in contrast to conventional treatment modalities.1,2 The main mechanism of PDT is that an employing photosensitizer (PS) upon irradiation could trigger a sequence of complicated photochemical and photobiological reactions to cause cancer cell apoptosis and necrosis by generating highly cytotoxic reactive oxygen species (ROS), particularly for singlet oxygen (1O2).3 To achieve the best performance for PDT, the selected PSs should have high quantum yields, large Stokes’ shifts, and long luminescent lifetimes in order to generate more ROS. Porphyrin and macrocyclic derivatives are widely used PSs, which play vital roles in many sophisticated biological processes and other artificial light harvesting systems.4,5 For PDT, self-assembly of those porphyrin derivatives into nanoscale structures or encapsulating into host materials or polymers represent three types of normal methods.6−11 However, the strong hydrophobic interactions and π−π stacking of porphyrin itself make them easier to form larger aggregates in aqueous media and hamper the practical PDT application of those above porphyrin-based systems. The above limitations can be overcome by choosing porphyrin nanocarriers or adopting some new molecular assembly methods. Similar to metal−organic frameworks (MOFs),12,13 porous organic polymers (POPs),14−16 also named conjugated microporous polymers (CMPs), covalent organic frameworks (COFs), and microporous organic networks (MONs), are constructed by strong covalent bonds and have attracted much attention due to their regular pore © 2017 American Chemical Society

structures, tunable chemical compositions, and some potential applications in gas storage and separation,17−19 catalysis,20−23 and sensors.24,25 The above porous materials have been demonstrated as available candidates for introduction of some photoactive molecular blocks into frameworks or cavities, since its periodic structures could increase the molecular distance between two dyes, dilute the local concentrations of dyes, and avoid some unwanted behaviors, such as aggregated luminescence quenching. In contrast to well-documented nanoscale metal−organic frameworks (NMOFs) in biomedical applications,26−33 the POPs and its corresponding materials are only in their initial stage and few works have been reported.34−36 Recently, two 3D polyimide POPs with dia topology have been prepared for ibuprofen (IBU) delivery in an aqueous medium.37 However, for the further investigations whether in vitro or in vivo, the most important question is the hydrophobic nature and large size distribution; therefore, some necessary chemical modifications or developments of effective synthesized methods are imperative in order to be sure of the cellular uptake.38 The template strategy and the surfactant assisted emulsion approach have previously been proven to obtain and control the morphology and size of POPs. Notably, NMOFs have been utilized as self-templates to design and synthesize MOF−POP nanocomposites with enhanced gas separation.39−42 The superiorities of NMOF self-templates as compared with classical hard templates are the tunable functional sites, the high Brunauer−Emmett−Teller (BET) Received: January 18, 2017 Revised: February 24, 2017 Published: February 26, 2017 2374

DOI: 10.1021/acs.chemmater.7b00228 Chem. Mater. 2017, 29, 2374−2381

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Chemistry of Materials Scheme 1. Schematic of the Light-Activated UiO-AM@POP (UNM) Systema

a

(a) Synthesis and preparation of UNM nanoparticles. (b) Structure of POP on UNM nanoparticles. (c) The cellular uptake and photodynamic therapy in cells.

molar ratio of 2-amino-1,4-benzenedicarboxylic acid (NH2BDC) and benzene-1,4-dicarboxylic acid (BDC) is 1:1 after 24 h of exchange. To synthesize core−shell nanocomposites, a modified method by using mixed solvents of methanol (CH3OH) and trichloromethane (CHCl3) is used after considering the nature of porphyrin and the formation of imine bonds (see the Supporting Information). Core−shell MOF@POP composites are prepared by polymerization of H2P and terephthalaldehyde at different feeding ratios in the presence of UiO-AM. The UNM possesses optimal dispersibility when the mass ration of H2P and terephthalaldehyde to UiO-AM is 20% in the feed (Figure S1). Taking the sample with 20% feed as an example, the UNM with uniform element distributions shows nearly spherical shapes in contrast to the regular octahedral geometry of UiOAM in transmission electron microscopy (TEM) images (Figure 1a,b and Figure S2), and their surfaces from the SEM images become rough instead of the smooth surface of UiO-AM (Figure S3). This result validates the successful synthesis of UNM with a core−shell structure. The average size distribution of UNM dispersed in DMF is about 176 nm determined by dynamic light scattering (DLS), which is in agreement with the results of TEM images. The crystallizability and structural stability of UNM are evidenced by powder X-ray diffraction (PXRD). UiO-AM and UNM show identical Bragg diffraction peaks, indicating that the growing of POPs is amorphous which does not alter the parent crystalline structure of UiO-AM in the current experimental conditions (Figure 1c). The PXRD data also confirmed that UNM could exhibit favorable stability in a physiological environment, which is important for their application in biology (Figure S4). After POP formations, the color of the samples changes from pale

surface, and the pores or cavities existing on the outer surface of NMOFs, which do not need any chemical postmodifications to enhance the interaction between the template itself and compositions of shell section and accelerate the formation of core−shell nanocomposites. Our groups have used the UiO-type NMOFs as selftemplates to synthesize the polyaniline−MOF nanohybrids for photothermal therapy and fluorescent MOF@POP nanocomposites for enhanced cellular uptake.43,44 On the other hand, pioneering work from the group of Jiang has reported amine-based porphyrin molecules, tetrakis(4-aminophenyl)21H,23H-porphine (H2P), as building blocks to covalently connect with various function-substituted terephthalaldehydes to construct a series of imine-based POPs with regular twodimensional (2-D) networks. The pore engineering, electronic behaviors, and ROS dependent catalysis have also been investigated in detail.45−47 Following the above works, herein, we try to synthesize the photoactive porphyrin−POP−MOF nanocomposites (UNM) by the self-template strategy. The reported 2-D POPs formed by H2P and terephthalaldehyde have been selected as the model structures for a “proof of principle”. The singlet oxygen generation and photoinduced apoptosis of as-synthesized nanocomposites are studied at length (Scheme 1).

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RESULTS AND DISCUSSION The physicochemical properties of the NMOFs used, such as functional site distribution, size, and morphology, have a direct influence on the final structure of core−shell composites. Highly crystallized and nanoscale UiO-AM was synthesized in our previous work.44 The average dimeter size of UiO-AM is 165 nm, the same as the presynthesized UiO-66, and the final 2375


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Chemistry of Materials

Figure 1. Characterizations of as-synthesized MOF@POP nanocomposites. TEM images and hydrodynamic size distribution (inset) of UiO-AM (a) and UNM (b). Scale bars, 200 nm. (c) PXRD patterns and their color changes (inset) of simulated UiO-66, UiO-AM, and UNM after POP coating. (d) Nitrogen adsorption isotherm of UiO-AM (red) and UNM (blue) at 77 K. (e) Normalized UV−vis absorption spectra of H2P (black), UiO-AM (red), and UNM (blue) dispersed in DMF at room temperature. (f) FT-IR spectrum of UiO-AM, H2P-POPs, and UNM, respectively. (g) Solid-state 13 C NMR spectra of UiO-AM (pale red) and UNM (blue). (h) TGA analysis of UiO-AM (red) and UNM (blue) under an air atmosphere.

yellow for UiO-AM to the final dark brown of the composites in the inserted figure of Figure 1c. To eliminate the posibility of adsorbed H2P molecules within the inner pore of UiO-AM, the adsorption and desorption experiments have been done and given the slight color changes of samples after full DMF washing (Figure S5). The permanent porosity and structural integrity of UNM have been investigated by nitrogen adsorption isotherm at 77 K. As shown in Figure 1d, the type II adsorption curve is observed before and after POP coating. Their BET surface areas slightly decrease from 1059 m2/g of UiO-AM to 974 m2/g of UNM. The average pore-size distributions of UiO-AM and UNM are around 2.5 and 5 Å (Figure S6). Both the results of color changes and gas adsoprtion behaviors verify the formation of POPs instead of simple inclusion/encapsulation of H2P within the UiO-AM pores. The core−shell structures are further confirmed by UV− vis, FL, FT-IR, and NMR. After POP coating, the assynthesized UNM composites exhibit similar UV and emission spectra to those of H2P molecules (typical Soret band at 440 nm and three less intense Q bands in the range 500−700 nm) (Figure 1e and Figure S7a). Two new stretching vibration bands of CN at 1250 cm−1 and CN at 1614 cm−1 are observed in FT-IR spectra (Figure 1f). The absorbance band at 1700 cm−1 is belonging to the residual aldehyde group on the surface of UNM nanoparticles. The solid 13C NMR of the UNM sample (Figure 1g) exhibits a new peak at 178 ppm of CN, which indicates the successful formation of imine

bonds.48,49 Thermogravimetric analysis (TGA) shows that both UiO-AM and UNM are stable up to about 250 °C and the content of POPs is about 8 wt % (Figure 1h). Considering the wide absorbance of porphyrin, three kinds of ROS indicators, such as indocyanine green (ICG),50 1,3diphenyl-isobenzofuran (DPBF),51,52 and 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA),53,54 have been selected to study the singlet oxygen generation ability of UNM nanoparticles under different irradiation wavelengths and their absorbance is further monitored by UV−vis absorption spectra. Irradiation of a solution of ICG in the presence of UNM (4 μg/ mL) causes a steady generation of singlet oxygen with a 450 nm laser source, as shown in the decreasing absorption band of ICG at 778 nm (Figure 2a). On the contrary, negligible spectra changes have been observed in control experiments, including the ICG only and UiO-AM and UNM nanoparticles in the dark (Figure 2b). Decrease of ICG absorption at 778 nm was inhibited significantly after addition of vitamin C as a ROS scavenger,55 further confirming the singlet oxygen generation upon light irradiation. Considering the Q band adsorption, the singlet oxygen efficacy of UNM (Figure S7b,c) is further evidenced by using DPBF as a detector under irradiation at 650 nm. Under the continuous red laser irradiation for 1 h in DMF, a negligible change of UV−vis absorption intensity was seen, indicating the good photostability of UNM (Figure S7d). On the other hand, intracellular singlet oxygen generation in cervical cancer cells (HeLa cells) was further investigated by 2376


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°C for 30 min. As shown in Figure 3c, UNM nanoparticles mainly locate within the endosome and the colocalization of UNM (green) with the endosome (red) produces an orange fluorescence in the merged images. These results confirmed UNM could be internalized effectively by cancer cells. The phototoxicities against human hepatocellular carcinoma (HepG2) and HeLa cells were investigated by using standard thiazolyblue tetrazolium bromide (MTT) assay. No obvious cellular death is observed in the absence of light irradiation even at a concentration of 25 μg/mL after 48 h of incubation, indicating the good biocompatibility of both as-synthesized UNM and UiO-AM (Figure 4a,b and Figure S8). Under irradiation with light at 450 nm, a dose-dependent cytotoxicity of UNM nanoparticles is observed in both HepG2 and HeLa cells. UNM nanoparticles also exhibit time- and powerdependent phototoxicities, and their corresponding halfmaximal inhibitory concentration (IC50) against HepG2 and HeLa is listed in Table 1. The IC50 value against HeLa cells is 2.1 μg/mL after 15 min of irradiation under 45 mW/cm2 laser intensity. Moreover, the anticancer efficiency of UNM nanoparticles was examined by the live/dead staining (Figure S9). Almost all of the HeLa cells are dead after being incubated with the UNM nanoparticles under irradiation, which is in agreement with the above MTT results. To further investigate the cell death mechanism, HeLa cells are double-labeled with Annexin V-FITC (fluoresceine isothiocyanate) and PI (propidium iodide) before flow cytometry analysis. In contrast to control groups (Figure 3c−e), an obvious increasing apoptotic rate (Q2 + Q3) is present in the UNM nanoparticle experimental group in all examined irradiation times (Figure 3f−h). The ratios of apoptosis cells are 52.6% for 5 min, 61% for 10 min, and 80.7% for 15 min, respectively, validating the irradiation time-dependent anticancer efficacy of UNM nanoparticles.

Figure 2. Singlet oxygen generation ability of as-synthesized MOF@ POP nanocomposites. (a) Time-dependent UV absorption spectra of ICG at 778 nm with UNM in PBS after irradiation with a 450 nm lamp from 0 to 180 s. (b) Comparison of the decay rate of ICG alone (black), UiO-AM (red), UNM in the dark (blue), and UNM upon irradiation with (green) or without (pink) the vitamin C scavenger, respectively. (c) The generation of intracellular ROS mediated by UNM samples upon light irradiation of 10 mW/cm2 for 5 min indicated by the fluorescence of DCF about the blank control, UNM pretreated with light, and UNM upon irradiation with or without vitamin C treatment. Scale bars, 20 μm.

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CONCLUSIONS In summary, a self-template synthesis approach has been applied to the design and preparation of photoactive MOF@ POP nanocomposites. The size distribution and morphology of such core−shell nanoparticles could be simply controlled by tuning the amine sites on the outer surface of selected UiO-AM seeds and the feeding ratios of H2P and terephthalaldehyde. After POP chemical modification, the high crystallization, pore integrity, and well-dispersed size distribution of UNM nanoparticles maintain well as UiO-AM itself. Importantly, the above light-activated system exhibits an efficient ability to generate singlet oxygen under various experimental conditions and PDT efficacy by photoinduced apoptosis of cancer cells. We believe that this template-derived microporous organic material approach may open a new way for formulating nanoscale organic polymers and expanding their applications in biomedical fields.

using DCFH-DA as indicators under confocal laser scanning microscopy (CLSM). As shown in Figure 2c, negligible green fluorescence is observed in all control groups, because there is no adequate singlet oxygen to oxidize nonfluorescent DCFHDA into fluorescent dichlorofluorescein (DCF). In contrast, a bright green fluorescence appears in UNM samples under light irradiation due to producing singlet oxygen. This result is further confirmed in the presence of ROS scavenger-vitamin C. Hence, such as-synthesized UNM nanoparticles can be potentially applied as PDT agents for effective killing of cancer cells. The cellular uptake of MOFs@POPs in HeLa cells was investigated by using CLSM and flow cytometry. The cell nuclei are stained with 4,6-diamidino-2-phenylindole (DAPI). CLSM images and flow cytometry (Figure 3a) show an enhanced intracellular distribution of UNM in HeLa cells with prolonged incubation time from 2 to 6 h. The uptake of UNM increased dramatically when the temperature reached 37 °C from 4 °C, indicating the ATP-mediated endocytosis (Figure 3b).56,57 Fluorescence colocalization analyses were carried out by using Lyso-tracker Red.58,59 HeLa cells were first incubated with UNM for 1 h and then stained with Lyso-tracker Red at 37

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EXPERIMENTAL SECTION

Materials and Physical Measurements. All of the starting materials were obtained commercially and were used without further purification. Anhydrous N,N-dimethylformamide (DMF) was stored over activated molecular sieves and was distilled under reduced pressure. UiO-66 and UiO-AM are synthesized by our previous works.44,60 All of the other solvents were purified according to the standard methods whenever needed. Fourier transform infrared (FT-IR) spectra were recorded on a Nicolet Impact 410 spectrometer. The solid-state 13C NMR spectra 2377


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Figure 3. Cellular uptake of MOF@POP nanocomposites. (a) Representative confocal laser scanning microscopy (CLSM) images of HeLa cells incubated with UNM nanoparticles at 4 and 37 °C for 0.5 h. (b) CLSM images, flow cytometry analysis, and mean fluorescence intensity of HeLa cells incubated with UNM nanoparticles for 2, 4, and 6 h, respectively. (c) CLSM images of HeLa cells induced by UNM nanoparticles in the presence of Lyso-tracker Red. Scale bar, 20 μm.

Figure 4. In vitro photocytotoxicities of MOF@POP nanocomposites at different laser intensities, irradiation times, and PS concentrations against HepG2 cells (a) and HeLa cells (b). Flow cytometry analysis of HeLa cells incubated with blank (c), only irradiation (d), and UNM without irradiation (e) and with different irradiation times of 5, 10, and 15 min (f−h). The four areas represent the different phases of the cells: necrotic (Q1), late-stage apoptotic (Q2), early apoptotic (Q3), and live (Q4).

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incubated for 40 min. Under the same conditions, VC (200 × 10−6 M) was used as the negative control. Light irradiation was used subsequently (10 mW/cm2 for 5 min). The outcomes were observed with CLSM as soon as possible (excitation wavelength, 488 nm; emission band-pass, 500−550 nm). Intracellular Uptake Studies. To verify the cellular uptake of UNM nanoparticles, HeLa cells were seeded in 6-well plates at about 200,000 cells/well in 2 mL of Dulbecco Modified Eagle Medium (DMEM, GIBCO) containing 10% fetal bovine serum, supplemented with 100 U/mL penicillin and 100 U/mL streptomycin, and incubated at 37 °C in a 5% CO2 atmosphere for 24 h. Then, the cells were washed with PBS three times and incubated at 37 °C for another 2, 4, and 6 h with UNM nanoparticle treatment to evaluate the uptake. At last, the cells were counterstained with DAPI for the cell nucleus. The fixed cells were again washed with PBS 7.4 four times before observation with a confocal laser scanning microscope (Carl Zeiss LSM 780). For the flow cytometry analysis, HeLa cells were seeded on 6-well plates and incubated for 24 h and 25 μg/mL UNM nanoparticles were added. After being washed with PBS three times, the cells were digested with trypsin and centrifuged. The precipitated cells was collected and dispersed in 0.5 mL of PBS. The 675 nm channel was used to detect the fluorescence intensity. Fluorescence colocalization analyses were carried out by using Lyso-tracker Red. HeLa cells were first incubated with UNM nanoparticles for 1 h, then stained with Lyso-tracker Red (100 nM) at 37 °C for 30 min, and then washed with PBS three times before being imaged by CLSM. The excitation wavelength was 555 nm. In Vitro PDT Studies against HepG2 and HeLa Cells. HepG2 and HeLa cells harvested in a logarithmic growth phase were seeded in 96-well plates at a density of 8 × 103 cells/well and incubated in DMEM containing 10% fetal bovine serum for 24 h. The media were then replaced by culture medium containing UiO-AM or UNM nanoparticles with various concentrations (0, 5, 10, 15, 20, and 25 μg/ mL). At the sixth hour, the UiO-AM and UNM nanoparticle groups were irradiated by a LED lamp with a wavelength of 450 nm at 10, 30, and 45 mW/cm2 for 5, 10, and 15 min, respectively. The cells were incubated for another 48 h. Then, 20 μL of MTT solution with a concentration of 5 mg/mL was added and the plates were incubated for another 4 h at 37 °C, followed by removal of the culture medium containing MTT and addition of 150 μL of dimethyl sulfoxide (DMSO) to each well to dissolve the formazan crystals formed. Finally, the plates were shaken for 5 min, and the absorbance of formazan product was measured at 490 nm by a microplate reader.

Table 1. IC50 Values of UNM Nanocomposites against HepG2 and HeLa Cells IC50 (μg/mL) HepG2 HeLa

10 mW/cm2 a 30 mW/cm2 a 5 minb 10 minb 26.9 9.6

13 4.1

19.4 12.6

12.4 5.2

15 minb 9.4 2.1

a The irradiation time is 15 min. bThe irradiation intensity is 45 mW/ cm2.

were recorded at 5K Hz. PXRD was performed by a Riguku D/ MAX2550 diffractometer using Cu Kα radiation, 40 kV, 200 mA with a scanning rate of 0.4°/min. The thermogravimetric analysis (TGA) was performed using a NetzchSta 449c thermal analyzer system at a rate of 10 °C/min under an air atmosphere. 1H NMR spectra were recorded on a Bruker NMR-400 DRX spectrometer at room temperature. The mass spectra (MS) of the samples were recorded by a Bruker autoflex III smart beam MALDI-TOF/TOF mass spectrometer with a smart beam laser at a 355 nm wavelength. UV− vis absorption spectra were monitored with a Shimadzu UV-2450 PC UV/vis spectrophotometer. The fluorescence intensity tests were obtained using a PerkinElmer LS-55 spectrofluorophotometer. The morphology of the nanoparticles was measured by transmission electron microscopy (TEM) characterized by a JEOL JEM-1011 electron microscope operating at an acceleration voltage of 100 kV. Confocal laser scanning microscopy (CLSM) images were taken using a Zeiss LSM 700 (Zurich, Switzerland). The nitrogen adsorption isotherm was measured on a Micromeritics ASAP 2010 analyzer. Synthesis of MOF@POP (UNM) Nanoparticles. A 30 mL portion of anhydrous CH3OH and CHCl3 (v:v = 3:1) containing UiOAM (356 mg), H2P (47.39 mg, 0.0703 mmol), terephthalaldehyde (24.74 mg, 0.1846 mmol), and 50 μL of acetic acid was heated to 75 °C for 48 h. After cooling to room temperature, the resulting product was washed with anhydrous DMF and methanol until the supernatant was colorless. The last supernatant was collected to make sure that there were not H2P molecules through UV−vis detection. The ultimate UNM nanoparticles were obtained through centrifugation (12,000 rpm × 10 min) and dried under a vacuum for 48 h at 120 °C to clear away the toxic organic solvent. Percentages of 30 and 40% for H2P and terephthalaldehyde feeding ratios were also synthesized under the same conditions. Singlet-Oxygen Generation Measurements. In vitro singlet oxygen detection of UNM nanoparticles was carried out by a modified method using indocyanine green (ICG) and DPBF as capture agents. An amount of 2 mL of H2O containing ICG (14 mg) and 8 μg of photosensitizer sample in a quartz cuvette was illuminated by the LED lamp (power density of 30 mW/cm2 at a wavelength of 450 nm) at room temperature for 300 s. The absorbance intensity of ICG at the maximum wavelength of 778 nm was detected for 0 to 180 s. The rate of singlet oxygen generation was determined from the reduced absorbance intensity over time. For the control experiments, ICG absorption with and without UiO-AM was also recorded under the same conditions. We detected the ICG absorption with UNM nanoparticles under dark conditions as the control. VC (50 × 10−6 M, an ROS scavenger) added to slow down the quenching speed of ICG was used as the negative control. We also employed the DPBF in DMF to evaluate the singlet oxygen generation ability of UNM nanoparticles. In the specific detection, UNM nanoparticles (4 μg/ mL) and DPBF (0.04 μM) were dissolved in DMF and the same time intervals as mentioned before were employed. A laser with a wavelength of 650 nm at an intensity of 200 mW/cm2 was used to irradiate the solution. The 650 nm laser was also used to irradiate the solution of UNM nanoparticles in PBS, and the absorbance intensity was recorded every 10 min. Intracellular ROS Detection. The CLSM observation method was also employed to detect the ROS generation. First, HeLa cells were incubated with UNM nanoparticles for 6 h, and then, the culture medium was replaced and washed three times. Then, the DMEM containing DCFH-DA (10 × 10−6 M) was added and further

Cell Viability (%) = [(A s − A b)/(Ac − A b)] × 100% As is the OD value of the treatment group, Ac is the OD value of the control group, and Ab is the OD value of the blank well. Each independent experiment was performed in triplicate. Calcein-AM/PI Test. To visually demonstrate the efficiency of UNM nanoparticles in photodynamic therapy, the HeLa cells were stained with propidium iodide and calcein-AM to identify dead (red) and live (green) cells, respectively. The control and drug-treated cells were incubated at 37 °C for 24 h in a humidified atmosphere containing 5% CO2. Then, all dishes of cells were irradiated for 5, 10, and 15 min with an LED light with a wavelength of 450 nm at an intensity of 45 mW/cm2. Then, the cells were further incubated at 37 °C for another 48 h. After staining with calcein-AM/PI for 40 min and washing with PBS, the two samples of cells were imaged with a fluorescence microscope. Cell Apoptosis and Necrosis by Annexin V Staining. The apoptosis and necrosis induced by PDT of UNM nanoparticles were evaluated by flow cytometry. HeLa cells were seeded at 1 × 106 cells/ well in 6-well plates and further cultured for another 48 h. Then, the culture media were replaced by 1 mL of fresh culture media containing 10% FBS. UNM nanoparticles was added to the cells, at a concentration of 25 μg/mL. Cells incubated with PBS served as a blank control. After 6 h of incubation, the cells were irradiated with LED light (450 nm) at 45 mW/cm2 for 5, 10, and 15 min, respectively. Following incubation of 48 h, the floating dead cells and adherent alive cells were all collected and stained with propidium iodide (PI) (dead 2379


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cells) and an Annexin V-FITC (apoptosis cells) apoptosis detection kit (Beyotime Biotechnology) according to the manufacturer’s instructions. The apoptosis and necrosis results were examined on a flow cytometer.

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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/acs.chemmater.7b00228. TEM and SEM images, powder X-ray diffractions, pore size distributions, fluorescence and absorption spectra, relative cell viabilities, and fluorescence microscope images (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Lei Wang: 0000-0003-4395-5002 Zhigang Xie: 0000-0003-2974-1825 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support was kindly provided by the National Natural Science Foundation of China (Project No. 21401188 and 51522307)

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REFERENCES

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