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Sequential Protein-Responsive Nanophotosensitizer Complex for Enhancing Tumor-Specific Therapy Downloaded via GUILFORD COLG on July 18, 2019 at 02:54:02 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Xingshu Li,†,‡,⊥ Huanhuan Fan,†,⊥ Tian Guo,‡ Huarong Bai,† Nahyun Kwon,‡ Kwang H. Kim,§ Sungsook Yu,§ Yejin Cho,§ Hyunji Kim,§ Ki Taek Nam,*,§ Juyoung Yoon,*,‡ Xiao-Bing Zhang,*,† and Weihong Tan† †
Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082, China ‡ Department of Chemistry and Nano Science, Ewha Womans University, Seoul 03760, Republic of Korea § Severance Biomedical Science Institute, Brain Korea 21 PLUS Project for Medical Science, College of Medicine, Yonsei University, Seoul 03760, Republic of Korea S Supporting Information *
ABSTRACT: A major challenge in cancer treatment is the development of effective tumor-specific therapeutic methods that have minimal side effects. Recently, a photodynamic therapy (PDT) technique using activatable photosensitizers (aPSs) has shown great potential for cancer-specific treatment. Here, we develop a sequential protein-responsive aPS (PcC4-MSN-O1) that is based on zinc(II) phthalocyanine derivative (PcC4)-entrapped mesoporous silica nanoparticles (MSNs) and a wrapping DNA (O1) as a biogate. Inside the nanostructure of PcC4-MSNO1, PcC4 shows self-quenching photoactivity. However, when PcC4-MSN-O1 sequentially reacts with telomerase and albumin, its photoactivity is dramatically turned on. Therefore, PcC4-MSN-O1 displays selective phototoxicity against cancer cells (e.g., HeLa) over normal cells (e.g., HEK293). Following systemic PcC4-MSN-O1 administration, there is an obvious accumulation in HeLa tumors of xenograftbearing mice, and laser irradiation clearly induces the inhibition of tumor growth. Moreover, the time-modulated activation process in tumors and the relatively fast excretion of PcC4-MSN-O1 indicate its advantages in reducing potential side effects. KEYWORDS: nanophotosensitizer, photodynamic therapy, activatable, protein-responsive, phthalocyanine
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without the initiation of resistance, and providing an immune response.14−16 However, “always on” PSs are utilized in most PDT approaches.17 As a consequence, the typical PDT approach suffers the drawback that patients must stay in the dark for a long period (usually 4−6 weeks) after treatment so that the PSs can be excreted from the body. Otherwise, photosensitivity of the skin and damage to other tissues can occur.18 In addition, after systemic administration of “always on” PSs, the eyes of patients are sensitive to bright indoor/
lthough many therapeutic modalities have been widely explored in the past few decades, effective tumorspecific therapy with minimal side effects still remains a great challenge.1−6 Owing to its spatiotemporal selectivity, photodynamic therapy (PDT) has shown great potential in tumor-specific therapy.7−10 In combination with light and molecular oxygen, photosensitizers (PSs), the substances administered during PDT, generate reactive oxygen species (ROS) to destroy tumor tissues and cells. PDT exhibits spatiotemporal selectivity because ROS generation occurs in only the region exposed to light, and the consequent chemical processes take place within approximately 50 nm of this location.11−13 In addition to this inherent selectivity, PDT has shown the advantages of being noninvasive, having a relatively wide-spectrum anticancer effect, allowing repeated treatments © 2019 American Chemical Society
Received: February 8, 2019 Accepted: June 4, 2019 Published: June 4, 2019 6702
DOI: 10.1021/acsnano.9b01100 ACS Nano 2019, 13, 6702−6710
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Scheme 1. Schematic Illustration of Dual Protein-Driven, Sequential Responsive Processes as Well as the Activatable Photoactivity of PcC4-MSN-O1a
FL, fluorescence; ROS, reactive oxygen species; MSN, mesoporous silica nanoparticle.
a
Figure 1. Albumin-responsive photoactivity of PcC4. (a) Absorption and (b) fluorescence spectra (excited at 630 nm) of PcC4 (2 μM) in water with and without HSA (1 mg/mL); F.I., fluorescence intensity. (c) ROS generation of PcC4 (2 μM) in water with and without HSA (1 mg/mL), detected by using 2,7-dichlorofluorescein diacetate (12 μM) as a probe. (d) Fluorescence intensity (706 nm) responses of PcC4 to various proteins in water. One HSA; 2 IgG; 3 hemoglobin; 4 0.1% fetal bovine serum (FBS); 5 1% FBS; 6 10% FBS. (e) Proposed mechanism for the self-quenching and albumin-driven “turn on” photoactivity of PcC4.
developed an aPS based on “one protein target” (one key)driven disaggregation and activation for tumor-specific treatment.25 To further enhance the tumor specificity, we demonstrate herein a different activatable design based on a “two protein target” (two key)-driven strategy. As shown in Scheme 1, because the fluorescence emission and ROS generation of the designed aPS require the presence of two different protein targets (telomerase and albumin), this design probably has the ability to enhance tumor-specific imaging and
outdoor light, and their skin is easily sunburned, swollen, and blistered.19,20 A new PDT technique has been developed in recent years; this technique uses activatable PSs (aPSs), which are substances that remain in a passive state (locked, OFF) even with light exposure but can be activated (unlocked, ON) at target sites by tumor-associated stimuli (keys).14,21,22 Because aPSs can be selectively activated at the sites where treatment is desired, they rejuvenate the PDT field by endowing a high possibility of minimizing side effects.17,23,24 Very recently, we 6703
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Figure 2. Fabrication of PcC4-MSN-O1 and its dual protein-driven, sequential responsive properties. (a) Morphology and (b) size distribution of the as-prepared MSNs determined using transmission electron microscopy and dynamic light scattering, respectively. (c) Zeta potential of the as-prepared MSNs, MSN-NH2, and PcC4-MSN-O1 in water. (d) Fluorescence spectra (excited at 630 nm) of PcC4-MSN-O1 (10 μM in PcC4) in pure water (black), in water containing telomerase (green), or in water containing both telomerase and HSA (red). dNTPs are added together with telomerase. The incubation times of telomerase and HSA were 2 h and 10 min, respectively. (e) Fluorescence intensity (at 706 nm) of PcC4-MSN-O1 incubated with telomerase for different times. Before detection, the mixture was incubated with HSA for 10 min.
biocompatibility.40−42 In addition, wrapping DNA O1, which responds to telomerase, could seal the MSN delivery platforms via strong electrostatic interactions.43 To achieve a dual stimulus-driven, sequential responsive aPS, we encapsulated PcC4 into MSN and used O1 as a biogate. First, MSNs with particle sizes of approximately 50 nm were synthesized (Figure 2a,b) because this size range is highly preferred for tumor passive accumulation via the enhanced permeability and retention (EPR) effect.44 Second, the MSNs were modified with 3-amino-propyltriethoxysilane (APTS), which resulted in positively charged surfaces (MSN-NH2, Figure 2c). Finally, MSN-NH2 were loaded with PcC4 and subsequently sealed by the negatively charged O1 to form a PcC4-MSN-O1 nanosystem. In this nanosystem, the loading of PcC4 was calculated to be 77.6 nmol/mg MSN-NH2, and the adsorption amount of O1 was calculated to be 0.3 μmol/g PcC4-MSN (Figure S1). Due to the relatively high stability of the PcC4 intermolecular interaction, caused by the hydrophobic phthalocyanine macrocycle π system, the PcC4 in this nanosystem shows self-quenched photoactivity (Figure S2 and Figure 2d, black line). Interestingly, a significant change in PcC4 fluorescence emission was observed in only the case of PcC4-MSN-O1 incubated with both telomerase and HSA (Figure 2d,e and Figure S3). When PcC4-MSN-O1 first encounters telomerase, O1 could be extended in the presence of dNTPs and detached from MSNs due to the generation of a rigid hairpin-like DNA structure. As a result, entrapped PcC4 molecules could be released but still form aggregates in aqueous solutions. When these mixed solutions were further added to HSA, PcC4 aggregates were disrupted due to the strong binding between the PcC4 monomer and albumin, leading to the recovery of photoactivity. It is worth noting that telomerase is an attractive cancer target because it is required in essentially all tumors for maintaining the length of telomeres and unlimited cell proliferation.45 In addition, many studies have claimed that albumin is most likely involved in the provision of nutrition to tumors.46,47 Therefore, these results
therapy. Below, we introduce how this design can realize the “sequential protein-responsive” concept.
RESULTS AND DISCUSSION Design and Fabrication of the Sequential ProteinResponsive aPS. In recent years, several porphyrin derivatives have been used as basic PSs to construct aPSs that respond to tumor-associated stimuli, such as the presence of folate receptors, hyaluronidase, or adenosine triphosphate, slightly acidic conditions, or redox changes.21,22,26−28 Compared to porphyrins, zinc(II) phthalocyanines generally show stronger absorption in the far-red/near-infrared region and a higher efficiency of generating ROS; thus, zinc(II) phthalocyanines provide higher potential for PDT application.29−37 In addition, two recent studies reported that zinc(II) phthalocyanines substituted with carboxyl groups strongly interact with albumin and consequently show controllable photoactivities.38,39 Hence, zinc(II) phthalocyanine tetra-α-substituted with 4-carboxylphenoxy groups (PcC4; see Scheme 1) was chosen as the model PS. As expected, PcC4 has excellent albumin-responsive photoactivity. The optical spectrum (Figure 1a,b) and ROS detection (Figure 1c) of PcC4 samples suggested that pure PcC4 forms H-type aggregates that exhibit highly quenched fluorescence and very limited ROS generation in water. However, the absorbance band at 700 nm, which indicates a monomer, increases in intensity upon the addition of human serum albumin (HSA). Additionally, fluorescence emission and ROS generation are dramatically recovered upon HSA addition. Moreover, significant changes in PcC4 fluorescence emission were not observed in the presence of other proteins (Figure 1d), indicating the selectivity of PcC4 for albumin. Based on these results, we speculate that albumin could induce the disaggregation of the PcC4 aggregates and then “turn on” PcC4 photoactivity (Figure 1e). Mesoporous silica nanoparticles (MSNs) have shown attractive ability to construct delivery platforms because of their large pore volumes, ease of surface functionalization, and 6704
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Figure 3. Selective phototoxicity of PcC4-MSN-O1 on cancer cells over normal cells. Cytotoxic effects of (a) HeLa cells and (b) HEK-293 cells incubated with different concentrations of PcC4-MSN-O1 or PcC4 alone for 3 h with white light irradiation (4100 K, 32 W, USA, 3 h). The data are expressed as the mean ± standard deviation (n = 5).
Figure 4. Tumor-specific imaging and PDT of PcC4-MSN-O1 after intratumoral injection. (a) In vivo fluorescence images of tumor-bearing mice before and after intratumoral injection of PcC4 alone or PcC4-MSN-O1 (50 μL, 100 μM). The red arrows indicate tumor sites. Fluorescence images were excited at 640 nm with an IVIS Lumina II imaging system. (b) Tumor growth of mice after saline (as control), PcC4, or PcC4-MSN-O1 treatment. At 4 h after treatment (100 μM each, intratumoral injection), the tumor was laser-irradiated at 660 nm (0.5 W/cm2 for 30 min). Tumor volumes were compared to their initial values. Data are expressed as the mean ± SEM (n = 5). (c) Average tumor weights on day 12 after different treatments. Data are expressed as the mean ± SEM (n = 5); ** indicates P < 0.01. (d) Histological analysis of tumors acquired from mice bearing tumors on day 12 after different treatments. Scale bar = 200 μm.
suggest that PcC4-MSN-O1 should have high potential for tumor-specific PDT. In Vitro Selectivity of PDT Effect of PcC4-MSN-O1 on Cancer Cells over Normal Cells. To evaluate the in vitro PDT effect of PcC4-MSN-O1, we first confirmed that the
cytotoxicity of MSNs was negligible (Figure S4a). Additionally, PcC4-loaded MSNs were not cytotoxic against HeLa cells without light irradiation (Figure S4b). Next, the cytotoxicity of PcC4-MSN-O1 upon illumination with white light (4100 K, 32 W, USA, 3 h) was tested. As shown in Figure 3a, PcC4-MSN6705
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Figure 5. Biodistribution, tumor-targeted PDT, and biocompatibility of PcC4-MSN-O1 after intravenous injection. (a) In vivo and (b) ex vivo fluorescence images of HeLa tumor-bearing mice before and after intravenous injection of PcC4-MSN-O1 (200 μL, 200 μM). The red circles indicate tumor sites. Fluorescence images were generated by exciting samples at 640 nm with an IVIS Lumina II imaging system. (c) Tumor growth of mice after saline (as control) or PcC4-MSN-O1 treatment. At 24 h after treatment (200 μM, intravenous injection), the tumor was laser-irradiated at 670 nm (0.5 W/cm2 for 20 min). Tumor volumes were compared to their initial values. Data are expressed as the mean ± SEM (n = 5). (d) Pathological analysis of organs acquired from mice bearing HeLa tumors on the day 13 after indicated treatments. Scale bar = 200 μm.
underwent a time-modulated activation process in tumors and relatively fast excretion from the body. In contrast, because PcC4 forms irregular aggregates of both small and large sizes in aqueous solutions (Figure S5), small parts of the PcC4 aggregates could be quickly activated by albumin in tumors, and then other parts of the PcC4 aggregates could undergo a slow activation process. It is also worth mentioning that PSs that are quickly excreted after PDT treatment are attractive because of their potential to reduce side effects. With these fluorescence imaging results, we then compared the PDT efficacy of PcC4-MSN-O1 to that of pure PcC4 after intratumoral injection and tumor-focused laser irradiation at 4 h postinjection. Changes in tumor volumes (Figure 4b) and weights (Figure 4c) and histological analysis (Figure 4d) of tumors after treatments indicated that PcC4-MSN-O1 has better PDT outcomes than PcC4. To further demonstrate the potential of PcC4-MSN-O1 for PDT, we next evaluated its biodistribution, in vivo PDT efficacy, and biocompatibility in tumor-bearing mice after intravenous injection. According to previous studies, after tail vein injection, a strong fluorescence signal is always observed throughout the whole body of the “always on” PS-administered mice, indicating the potential side effects of PSs, as we discussed in the introduction.20,33,34,48,49 As expected, PcC4MSN-O1 displayed a different phenomenon, with no obvious
O1 displayed concentration-dependent phototoxicity against HeLa cells (telomerase overexpressed). For example, 2.34 μM PcC4-MSN-O1 (the concentration of the entrapped PcC4 is indicated) could kill 90.55 ± 3.18% of HeLa cells. In contrast, PcC4-MSN-O1 did not show significant phototoxicity against normal cells (HEK-293) under the same conditions (Figure 3b). In addition, treatments with PcC4 alone did not show an obvious PDT effect on either HeLa or HEK-293 cells, which was probably because of its low intracellular delivery efficiency. These results indicate that PcC4-MSN-O1 has a selective PDT effect on cancer cells over normal cells. In Vivo PDT Efficacy of PcC4-MSN-O1 on TumorBearing Mice. Encouraged by the in vitro results, we then evaluated the in vivo PDT efficacy of PcC4-MSN-O1 on tumor-bearing mice. First, we detected the fluorescence change in tumor tissues after intratumoral injection with PcC4 alone or PcC4-MSN-O1. Compared to PcC4-injected tumors, PcC4MSN-O1-injected tumors showed lower fluorescence intensity in the first hour (Figure 4a). After 4 h, the fluorescence intensity in the PcC4-MSN-O1-injected tumors became approximately 2-fold that in PcC4-injected tumors. As time progressed, the fluorescence signals started to decline gradually in intensity. In addition, the PcC4-MSN-O1-injected tumor fluorescence decreased faster than PcC4-injected tumor fluorescence. These results suggested that PcC4-MSN-O1 6706
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ACS Nano fluorescence throughout the whole body (only at the live site, as an exception) after injection (Figure 5a,b). However, 4 h postinjection, tumor tissues started to display an obvious fluorescence signal, probably owing to the EPR effect and sequential protein-responsive activation. Importantly, nearly no fluorescence signal was observed in the whole body 2 days postinjection, which again indicated the relatively fast excretion of PcC4-MSN-O1. We also evaluated photodynamic antitumor effects of PcC4-MSN-O1 after intravenous injection. Compared to the control group (only laser irradiation), the group treated with PcC4-MSN-O1 exhibited a significant inhibition in tumor growth in the presence of 670 nm laser irradiation (Figure 5c). The excellent PDT outcome of PcC4-MSN-O1 was also confirmed by histological analysis (Figure S6). An increased number of cells with a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)positive signal indicated increased apoptosis in the group treated with PcC4-MSN-O1 and laser irradiation compared with the control group. Finally, to determine the biocompatibility of this treatment, several organs, including mouse heart, liver, lung, spleen, and kidney, were subjected to hematoxylin and eosin (H&E) staining on day 13 following treatment. The results showed that none of the mouse tissues treated with PcC4-MSN-O1 displayed pathological changes (Figure 5d). These results indicate that treating cancer with this sequential protein-responsive aPS is both effective and biocompatible.
Apparatus. Dynamic light scattering was measured on a Malvern Zetasizer Nano ZS90 (Malvern Instruments, Ltd., Worcestershire, UK). Transmission electron microscopy was carried out on an H7000 NAR transmission electron microscope (Hitachi) with a working voltage of 100 kV. All fluorescence measurements were carried out on a FluoroMax-4 spectrofluorometer (HORIBA JobinYvon, Edison, NJ) with a 200 μL quartz cuvette. UV−vis spectra were measured by a UV-2450 UV−vis spectrophotometer (Shimadzu). Synthesis of MSN. First, 1.17 g of CTAB was dissolved into 180 mL of water. Then, 30 mL of ethylene glycol and 7.20 mL of ammonia solution (25%) were introduced to the CTAB solution. The mixture was stirred strongly at 50 °C for 30 min. Then, 1.43 mL of TEOS and 0.26 mL of APTES were quickly added to the surfactant solution. The mixture was allowed to react for another 2 h under vigorous stirring. Then, the reaction solution was kept at 50 °C for 20 h without stirring. The resulting solid crude product was centrifuged, washed with deionized water and methanol, and dried in air to yield the as-synthesized mesoporous silica nanoparticles (denoted MSNs). To remove the surfactant template, 1 g of the as-synthesized MSNs was refluxed for 12 h in a methanolic solution of 0.3 g of NH4NO3 in 40 mL of methanol at 60 °C. The resulting product was centrifuged and extensively washed with deionized water and methanol. Synthesis of MSN-NH2. The surface of MSN was functionalized with amine groups by treatment with APTES. Fifty milligrams of MSN was dispersed in 50 mL of ethanol. The solution was then refluxed for 4 h, followed by the addition of 100 μL of APTES. After being centrifuged and washed with water, amine-functionalized MSNs were dried in air. Synthesis of PcC4-MSN. PcC4 (0.2 mg) and MSN-NH2 (2 mg) were added to 2 mL of solution (water/MeOH = 1:1). The mixture was ultrasonicated for 20 min. Then, the mixture was shaken (180 rpm/min) overnight at 37 °C and kept in the dark. Synthesis of PcC4-MSN-O1. One milliliter of PcC4-MSN was centrifuged and washed twice with diethyl pyrocarbonate (DEPC) water. Then, PcC4-MSN was suspended in 1 mL of DEPC water containing O1 (at a final concentration of 1 μM). The mixture was slightly shaken using a shaking table for 3 h at 4 °C before experiments. Cell Culture and Buffer. HeLa cells and HEK-293 cells were purchased from the ATCC (American Type Culture Collection, Manassas, VA, USA) and incubated in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) FBS (Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 °C in a 5% CO2 atmosphere. Dulbecco’s phosphate-buffered saline (DPBS) without Ca2+ and Mg2+ (Invitrogen) was used to wash cells. Cell Extracts. HeLa cells were collected in the exponential phase of growth, and 5 × 107 cells were dispensed in a 1.5 mL EP tube, washed twice with ice-cold DPBS, and resuspended in 200 μL of icecold 1xCHAPS lysis buffer. The mixture was incubated for 30 min on ice and centrifuged at 14 000 rpm at 4 °C for 20 min. The supernatant was collected as a cell extract for analysis or frozen at −80 °C. Sequential Protein Response Tests. To demonstrate the detachment of the O1 extension product from the MSNs in response to telomerase and HSA, cell extract and 5 μL of dNTPs (10 mM) were mixed with 200 μL of a PcC4-MSN-O1 probe (10 μM PcC4) in PBS buffer (pH 7.4, containing 136.7 mM NaCl, 2.7 mM KCl, 8.72 mM Na2HPO4, and 1.41 mM KH2PO4). After incubation at 37 °C for different periods of time, the supernatants were collected and incubated with HSA (1 mg/mL) for 10 min. The mixtures were irradiated with a FluoroMax-4 spectrofluorometer (HORIBA JobinYvon, Edison, NJ). ROS Detection. Detection of ROS was performed using 2,7dichlorofluorescein diacetate as the probe. In the presence of ROS, 2,7-dichlorofluorescein diacetate is oxidized to form dichlorofluorescein, which emits bright fluorescence around 520 nm. Briefly, photosensitizer (PcC4, 2 μM), HSA (1 mg/mL), and 2,7dichlorofluorescein diacetate (12 μM) were mixed in water. The mixtures were irradiated by using a 500 W halogen lamp and a water tank for cooling. After various irradiation times, fluorescence spectra
CONCLUSIONS In summary, we developed and successfully tested the dual protein-driven, sequential responsive strategy for the design of tumor-specific aPSs. The approximately 50 nm nanostructure and sequential telomerase/albumin-responsive photoactivity enable PcC4-MSN-O1 to display cancer cell-selective in vitro phototoxicity as well as time-modulated, tumor-specific, in vivo imaging-guided PDT. In addition, the fast excretion of PcC4MSN-O1 after PDT treatment further reduces its potential side effects. EXPERIMENTAL SECTION Materials and Reagents. Human serum albumin (HSA), IgG, hemoglobin, 2,7-dichlorofluorescein diacetate, and 1xCHAPS lysis buffer were purchased from Sigma-Aldrich Korea. dNTPs were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). PcC4 was prepared using a previously described procedure.50 Cetyltrimethylammonium bromide (CTAB), tetraethylorthosilicate (TEOS), aminopropyltriethoxysilane (APTES), and other chemicals were of analytical grade, purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China), and used without further purification. MTS (3-(4,5-dimethyl-thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was purchased from Promega Corporation. Hybridization buffer (pH 7.4) contained 20 mM Tris-HCl, 200 mM NaCl, and 8 mM MgCl2. All solutions were prepared using ultrapure water with an electrical resistance of >18.3 MΩ, which was prepared through a Millipore Milli-Q water purification system (Billerica, MA, USA). The DNA sequences in this work were synthesized and purified by Sangon Biotechnology (Shanghai, China) with the following sequences: Wrapping DNA (O1): 5′-(CCC TAA)6 AAT CCG TCG AGC AGA GTT-3′ FAM-O1:5′-FAM-CCC TAA (CCC TAA)5 AAT CCG TCG AGC AGA GTT-3′ Complementary-chain DNA (O2): 5′-AAC TCT GCT CGA CGG ATT (TTA GGG)6-3′ 6707
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ACS Nano (excited at 504 nm) of the mixtures were recorded using a RF-5301/ PC spectrofluorophotometer. Cytotoxicity of MSNs. HeLa cells (4 × 103) were seeded in a 96well plate and incubated for 24 h. After being washed twice with DPBS, the cells were treated with different concentrations of MSNs in culture medium supplemented with 10% FBS at 37 °C for 4 h. Then, the culture medium was replaced with 200 μL of fresh culture medium. After 48 h of incubation, the culture medium was discarded. The cells were washed with DPBS. Finally, HeLa cells were cultured in 100 μL of fresh culture medium and 20 μL of MTS reagent for a proliferation assay. After 30 min of incubation at 37 °C, cell viability was monitored by measuring the absorption at 490 nm using a Synergy 2 multi-mode microplate reader (Bio-Tek, Winooski, VT). In Vitro Phototherapeutic Efficacy Tests. HeLa cells or HEK293 cells were seeded in a 96-well plate at a density of 4 × 103 cells per well and incubated for 24 h. After the culture medium was removed, cells were incubated with different concentrations of PcC4 or PcC4-MSN-O1 at 37 °C for 4 h. Then, the culture medium was replaced with 200 μL of fresh culture medium. For PDT, cells were irradiated with white light (4100 K, 32 W, USA) for 3 h. After 48 h of incubation, the cell medium was replaced with 100 μL of fresh culture medium and 20 μL of MTS solution. After 30 min of incubation, the therapeutic efficacy of PDT on HeLa cells was assayed by measuring the absorbance at 490 nm using a Synergy 2 multi-mode microplate Reader (Bio-Tek, Winooski, VT). In Vivo Fluorescence Imaging after Intratumoral Injection. Female Balb/c mice (∼20 g) were obtained from the Hunan SJA Laboratory Animal Co., Ltd. and used under protocols approved by Hunan University Laboratory Animal Center. To generate a 4T1 tumor model, ∼5 × 106 4T1 cells in ∼100 μL of DPBS were subcutaneously injected into the shoulder of each mouse. The mice were treated when the tumor volumes approached 60−70 mm3. When the tumor size reached ∼60 mm3, 50 μL of PcC4 or PcC4-MSN-O1 (corresponding to 100 μM PcC4) was intratumorally injected into the tumor-bearing mice. Imaging was carried out using a VIS Lumina XR at different times after injection. In Vivo Phototherapeutic Efficacy Tests after Intratumoral Injection. Female Balb/c mice (∼20 g) were obtained from the Hunan SJA Laboratory Animal Co., Ltd. and used under protocols approved by Hunan University Laboratory Animal Center. To generate a 4T1 tumor model, ∼5 × 106 4T1 cells in ∼100 μL of DPBS were subcutaneously injected into the shoulder of each mouse. The mice were treated when the tumor volumes approached 60−70 mm3. When the tumor size reached ∼60 mm3, 4T1 tumor-bearing mice were divided into three groups of five animals/group to quantify the growth rate of tumors after the following treatments: (1) saline, (2) PcC4, and (3) PcC4-MSN-O1. The total amount of the reagent for each treatment was 50 μL. The PcC4 concentration was 100 μM. Four hours later, the tumors were exposed to a 660 nm laser (MDLMD-660 nm-17110106) with a power of 500 mW for 30 min. The cross section of the laser beam was adjusted to cover the entire region of the tumor. The sizes of tumors were measured by calipers every other day after the treatment. The volume of tumor (V) was calculated by the following equation: V = AB2/2, where A and B are the long and short diameter (mm) of the tumor, respectively. The relative volume of tumors was evaluated by normalizing the measured values to their initial volumes. The mice were finally sacrificed to examine the histopathology of tumors by H&E staining. Slides were recorded using a Pannoramic MIDI digital slide scanner with a Zeiss plan-apochromat objective (20× magnification, 0.8 numerical aperture) and a Hitachi (HV-F22CL) 3CCD progressive scan color camera (resolution = 0.2325 μm/pixel). In Vivo/Ex Vivo Fluorescence Imaging after Intravenous Injection. All animal procedures were approved by the Institutional Animal Care Committee at Yonsei University. HeLa cells (approximately 2 × 107 cells/mouse) were subcutaneously injected into male NOD-SCID mice. When the tumor volumes reached approximately 100 mm3, PcC4-MSN-O1 (200 μL, 200 μM) was intravenously injected into the tails of mice. In vivo/ex vivo fluorescence images were captured at different time points after the
sample injection using an animal optical imaging system (IVIS, Caliper Life Sciences). The samples were excited at 640 nm. In Vivo Phototherapeutic Efficacy and Biocompatibility Tests after Intravenous Injection. HeLa cells (approximately 2 × 107 cells/mouse) were injected into NOD-SCID mice. When the tumor volumes reached approximately 100 mm3, the mice (five mice each group) were intravenously injected with PcC4-MSN-O1 (200 μM, 200 μL) or saline (200 μL). After 24 h, mice were irradiated with a 670 nm laser (0.5 W/cm2, 20 min). The tumor sizes were determined using a caliper and measured for a duration of 13 days. Thirteen days after treatment, the mice were sacrificed and the harvested tumors were fixed in 4% paraformaldehyde and then embedded in paraffin. To evaluate side effects in the lung, liver, heart, kidney, and spleen, morphological changes were determined for all mice by a pathologist. Sections (4 μm) were stained with H&E and observed under the microscope (Olympus BX-43). For apoptosis analysis, sections (4 μm) were subjected to a TUNEL assay (click-iT Plus TUNEL assay, Invitrogen) following the manufacturer’s instructions. The numbers of TUNEL-positive cells (green signal) were measured in four selected microscopic fields (400×) using ImageJ software for three independent experiments.
ASSOCIATED CONTENT S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.9b01100. Figures S1−S6 (PDF)
AUTHOR INFORMATION Corresponding Authors
*E-mail:
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[email protected]. ORCID
Juyoung Yoon: 0000-0002-1728-3970 Xiao-Bing Zhang: 0000-0002-4010-0028 Weihong Tan: 0000-0002-8066-1524 Author Contributions ⊥
X.L. and H.F. contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS X.-B.Z. thanks the National Natural Science Foundation of China (Grant No. 21521063) and the Science and Technology Project of Hunan Province (2016RS2009 and 2016WK2002). J.Y. thanks the National Research Foundation of Korea (NRF), which was funded by the Korean government (MSIP) (Grant No. 2012R1A3A2048814). H.F. thanks the National Postdoctoral Program for Innovative Talents (BX201700072). K.T.N. thanks the Korea Mouse Phenotyping Project (NRF2016M3A9D5A01952416) of the National Research Foundation. REFERENCES (1) Sawyers, C. Targeted Cancer Therapy. Nature 2004, 432, 294− 297. (2) Benson, J. D.; Chen, Y. -N. P.; Cornell-Kennon, S. A.; Dorsch, M.; Kim, S.; Leszczyniecka, M.; Sellers, W. R.; Lengauer, C. Validating Cancer Drug Targets. Nature 2006, 441, 451−456. (3) Ferrari, M. Cancer Nanotechnology: Opportumities and Challenges. Nat. Rev. Cancer 2005, 5, 161−171. 6708
DOI: 10.1021/acsnano.9b01100 ACS Nano 2019, 13, 6702−6710
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DOI: 10.1021/acsnano.9b01100 ACS Nano 2019, 13, 6702−6710