pH-Responsive Dimeric Zinc(II) Phthalocyanine in Mesoporous Silica

Article

pH-Responsive Dimeric Zinc(II) Phthalocyanine in Mesoporous Silica Nanoparticles as an Activatable Nanophotosensitizing System for Photodynamic Therapy Roy Wong, Sun Y. S. Chow, Shirui Zhao, Wing-Ping Fong, Dennis K. P. Ng, and Pui-Chi Lo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06353 • Publication Date (Web): 29 Jun 2017 Downloaded from http://pubs.acs.org on July 1, 2017

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ACS Applied Materials & Interfaces

pH-Responsive Dimeric Zinc(II) Phthalocyanine in Mesoporous Silica Nanoparticles as an Activatable Nanophotosensitizing System for Photodynamic Therapy

Roy C. H. Wong,† Sun Y. S. Chow,† Shirui Zhao,† Wing-Ping Fong,‡ Dennis K. P. Ng,*,† and Pui-Chi Lo*,§

†

‡

Department of Chemistry and School of Life Sciences, The Chinese University of Hong §

Kong, Shatin, N.T., Hong Kong, China, and Department of Biomedical Sciences, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China

KEYWORDS: activatable, mesoporous silica nanoparticle, photodynamic therapy, photosensitizer, phthalocyanine, self-quenching

1 Environment ACS Paragon Plus


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ABSTRACT: An acid-cleavable acetal-linked zinc(II) phthalocyanine dimer with an azido terminal group (cPc) was prepared and conjugated to alkyne-modified mesoporous silica nanoparticles via copper(I)-catalyzed alkyne-azide cycloaddition reaction. For comparison, an amine-linked analogue (nPc) was also prepared as a non-acid-cleavable counterpart. These dimeric phthalocyanines were significantly self-quenched due to the close proximity of the phthalocyanine units inside the mesopores, resulting in much weaker fluorescence emission and singlet oxygen generation, both in N,N-dimethylformamide and in phosphate buffered saline (PBS), compared with the free molecular counterparts. Under acidic conditions in PBS, the cPc-encapsulated nanosystem was activated in terms of fluorescence emission and singlet oxygen production. After internalization into human colon adenocarcinoma HT29 cells, it exhibited much higher intracellular fluorescence and photocytotoxicity compared to the nanosystem entrapped with nPc. The activation of this nanosystem was also demonstrated in tumor-bearing nude mice. The intratumoral fluorescence intensity increased gradually over 24 h, while for the nPc counterpart the fluorescence remained very weak. The results suggest that this nanosystem serves as a promising activatable nanophotosensitizing agent for photodynamic therapy.


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1. INTRODUCTION Photodynamic therapy (PDT) is a relatively noninvasive therapeutic method for the treatment of cancer.1 Through the interaction of photosensitizers, light, and tissue oxygen, singlet oxygen (1O2) can be generated to kill neighboring cells and tissues. Much research effort has been devoted to developing advanced photosensitizing systems that can achieve tumor specificity, improved efficacy, and less side effects.2 The emergence of activatable photosensitizers in the past decade has shed light on the direction of further development of more advanced photosensitizers.3 A common design of these “smart” photosensitizers involves a detachable quencher or another photosensitizer moiety for self-quenching. The photosensitizing properties of these photosensitizers are quenched in their native state, but can be activated upon interaction with some cancer-related stimuli, such as mRNA,4,5 enzymes,6,7 intracellular thiols,8,9 or the acidic tumor microenvironment,10,11 which cleave the linkers and free the photosensitizer units. Recently, many nanosystems, such as liposomes, nanogels, polymeric micelles, and gold nanoparticles have been exploited to deliver molecular photosensitizers in order to enhance their therapeutic efficacy.12,13 The advantages of these nanocarriers include high photosensitizer payload, reduced non-specific localization of photosensitizers due to binding with plasma components, prevention of degradation of photosensitizers, and the enhanced permeability and retention effect. Apart from these nanocarriers, mesoporous silica nanoparticles (MSNs) are also a common drug delivery platform, which are generally stable toward microbials, enzymatic degradation, and environmental changes such as fluctuation in pH and temperature.14 Their inert nature can also tolerate a wide range of reaction conditions for chemical modifications. Therefore, MSNs are promising nanocarriers for photosensitizers and the encapsulation of activatable photosensitizers into MSNs could synergize the therapeutic effects for advanced PDT. However, to our knowledge, only a few activatable



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MSN-based nanophotosensitizing systems have been reported so far that are triggered by acid,15,16 thiols,17 or near-infrared light.18 Over the last few years, we have been interested in the development of molecular-based activatable photosensitizers that can be triggered by acids and/or thiols.19-22 For the selfquenched phthalocyanine systems,19,22 it has been found that the pH-responsive tetramer exhibits a stronger self-quenching effect and a more remarkable activatable response than the dimeric counterpart. While the in vitro photocytotoxicity of the non-cleavable dimeric analogue is only slightly lower than that of the cleavable dimer, the non-cleavable tetrameric analogue remains non-cytotoxic even in the presence of light. The results imply that the native form of the acid-cleavable tetramer would also be non-cytotoxic in a non-acidic environment. This behavior is desirable and may render the photosensitizer exhibiting significantly different toxicity toward the healthy tissues and the generally acidic tumor microenvironment. As an extension of these studies, we report herein a nanophotosensitizing system in which a pH-responsive phthalocyanine dimer is conjugated to MSNs. Owing to the close proximity of the phthalocyanine rings inside the mesopores, their fluorescence as well as singlet oxygen production are largely quenched in the native state and effectively restored upon acidic treatment or internalization into cancer cells, rendering this nanosystem a promising theranostic agent for cancer. The synthesis, characterization, pH-responsive properties, as well as the in vitro and in vivo activation of this photosensitizing system are reported below.

2. RESULTS AND DISCUSSION 2.1. Molecular Design and Synthesis. Zinc(II) phthalocyanines (ZnPcs) are welldocumented as efficient photosensitizers,23 but their aggregates are self-quenched and become photodynamically inactive. On this basis, we employed an acetal-linked ZnPc dimer


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as a pH-responsive photosensitizer and conjugated it to MSNs. We hypothesized that the dimeric ZnPc molecules would pack closely inside the mesopores of MSNs, thereby promoting the self-quenching effect resulting in negligible fluorescence emission and singlet oxygen generation, and upon activation in acidic environments, the response of restoration of these properties would be prominent. The use of MSNs as a nanoplatform could also bring the aforementioned advantages, making the resulting nanoparticles a multifunctional photosensitizing system. Scheme 1 shows the synthetic route for the MSNs encapsulating the cleavable dimeric ZnPc cPc. The alkynyl ZnPc 1 was prepared according to our previously described procedure,24 while the linker 2 was synthesized by condensation reaction between 4-(2bromoethoxy)benzaldehyde and 3-azido-1-propanol. These two compounds then underwent copper-catalyzed azide-alkyne cycloaddition reaction to give dimeric ZnPc 3. It was then followed by nucleophilic substitution reaction with sodium azide to afford cPc. The MSN 4 was prepared according to literature procedure with minor modification.25,26 Propargyl groups were then introduced to the mesopores by treating 4 with propargyl bromide in the presence of K2CO3. Attempts to conjugate the resulting MSN 5 with cPc under the condition of CuSO4·5H2O and sodium ascorbate in CHCl3/EtOH/H2O (12:1:1, v/v) were not successful. Surprisingly, the reaction led to the isolation of a substantial amount of a monomeric ZnPc (mPc) which might be formed by hydrolysis of cPc (Scheme S1 in Supporting Information). This compound was then used as a reference compound in the subsequent studies. By changing the click reaction condition to CuI and Et3N in N,N-dimethylformamide (DMF), we were able to obtain the conjugate cPc@MSN, which was collected by centrifugation and then washed with sodium N,N-diethyldithiocarbamate and DMF to remove the adsorbed copper ions. For comparison, a non-cleavable dimeric phthalocyanine was also prepared in which the acetal linker of cPc was replaced with an amino group. This dimer nPc was then immobilized



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into MSN 5 with a similar procedure to afford nPc@MSN (Scheme S2 in Supporting Information).

Scheme 1. Synthesis of cPc@MSN.

2.2. Characterization and Photophysical Properties. Figure 1a shows the transmission electron microscopy (TEM) images of all the MSNs, including 4, 5, cPc@MSN, and nPc@MSN. All of them were spherical in shape with a diameter of ca. 100 nm and a porous structure. The size was typical for the MSNs prepared under similar conditions25 and the encapsulation of dimeric ZnPcs did not significantly change the dimension. The hydrodynamic radii of these nanoparticles were also measured in water using dynamic light scattering (Figure 1b). The values ranged from 121 nm (for 5) to 187 nm (for nPc@MSN). It seems that the MSNs showed slight aggregation in water, which is quite common for this kind of non-coated silica nanoparticles.27,28 The loading of dimeric ZnPc in cPc@MSN was determined by dispersing the nanoparticles in DMF with 1 mM HCl and then measuring the absorbance of the Q band of the released monomeric ZnPc at 690 nm, using mPc as the standard. The loading of


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nPc@MSN was estimated by comparing its Q-band absorbance with that of cPc@MSN, assuming that they have the same aggregation tendency. The loading was found to be 12% (by weight) for cPc@MSN and 8% (by weight) for nPc@MSN. (a)

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nPc@MSN

187 ± 30

Figure 1. (a) TEM images of (i) 4, (ii) 5, (iii) cPc@MSN, and (iv) nPc@MSN (scale bar =

100 nm). (b) Hydrodynamic radii of different MSNs measured by dynamic light scattering. To confirm the presence of primary amino groups in MSN 4 and the completion of the conversion from 4 to 5, a ninhydrin test was performed by which the presence of primary amines is indicated by an absorption band at 588 nm for the oxidized product.29 As shown in Figure S1 in Supporting Information, a strong band at 588 nm was observed for MSN 4, while this band completely vanished for MSN 5, indicating that virtually all the primary amino groups of 4 were consumed after the substitution with propargyl bromide. Attempts were also made to characterize the MSNs with FT-IR spectroscopy. As shown in Figure S2 in Supporting Information, all the spectra of 4, 5, cPc@MSN, and nPc@MSN showed the characteristic peaks of MSNs, namely Si-O-Si asymmetric stretch at ca. 1070 cm1

, Si-OH stretch at ca. 960 cm-1, and Si-O-Si symmetric stretch at ca. 800 cm-1.30 The strong

bands at ca. 3440 cm-1 and 1640 cm-1 could be assigned to the stretching and bending modes of the adsorbed water, respectively.31 A weak band at 2960 cm-1 was also observed as typical C-H stretches. Unfortunately, the absorptions due to the phthalocyanine rings (for cPc@MSN and nPc@MSN) could not be seen, which could be attributed to the low loading and the fact that the weak signals were masked by the strong bands of the MSN host and the adsorbed water molecules. The electronic absorption and fluorescence spectra of all the new phthalocyanines and their MSN conjugates were measured in DMF, and the data are summarized in Table 1. Figure 2a shows the electronic absorption spectra of the dimers cPc and nPc, as well as their MSN conjugates cPc@MSN and nPc@MSN in DMF. For comparison, the spectrum of the monomer mPc is also included. While the spectra of mPc, cPc, and nPc are typical as those


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for non-aggregated ZnPcs, the Q bands of cPc and nPc (at 1 µM) are slightly weaker than that of mPc (at 2 µM), which suggests that the dimeric ZnPcs are slightly aggregated. For the two dimeric ZnPc-encapsulated MSNs, the Q bands are much weaker and broader, indicating that the ZnPc rings are substantially aggregated inside the mesopores of the MSNs. This was supported by their different fluorescence intensities. As shown in Figure 2b and Table 1, the fluorescence of the dimeric cPc and nPc was significantly quenched when compared with that of mPc, and the fluorescence of the MSN conjugates cPc@MSN and nPc@MSN could hardly be seen. The results further suggest that the two ZnPc units in the dimers are selfquenched and by encapsulating these dimeric molecules into MSNs, the ZnPc moieties are even more aggregated and severely self-quenched in term of fluorescence emission. (a)

0.5 2 µM mPc 1 µM cPc 1 µM nPc cPc@MSN (with 1 µM cPc) nPc@MSN (with 1 µM nPc)

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2 µM mPc 1 µM cPc 1 µM nPc cPc@MSN (with 1 µM cPc) nPc@MSN (with 1 µM nPc)

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Figure 2. (a) Electronic absorption and (b) fluorescence spectra of different ZnPc systems in DMF. Table 1. Electronic absorption and photophysical data for mPc, cPc, nPc, cPc@MSN, and nPc@MSN in DMF.

a

ZnPc System

λabs /nm (log ε)

λexa /nm

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Φ∆c

mPc

343 (4.76), 622 (4.55), 690 (5.30)

708

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0.63

cPc

342 (5.11), 635 (4.95), 690 (5.51)

707

0.03

0.30

nPc

338 (4.93), 621 (4.76), 689 (5.51)

701

0.03

0.19

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340, 640, 692

-d

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0.01

nPc@MSN

339, 639, 691

-d

-d

0.03

Excited at 610 nm. b Relative to unsubstituted ZnPc in DMF as the reference (ΦF = 0.28). c

Relative to unsubstituted ZnPc in DMF as the reference (Φ∆ = 0.56). determined.


d

Could not be

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To evaluate the photosensitizing efficiency of these systems, their singlet oxygen generation efficiency in DMF was compared by using 1,3-diphenylisobenzofuran (DPBF) as the singlet oxygen scavenger. Figure 3 shows the rate of photodegradation of DPBF sensitized by these ZnPc systems. It can be seen that the generation of singlet oxygen by cPc and nPc was still substantial, though not as efficient as that of mPc. The generation of singlet oxygen was almost fully inhibited in cPc@MSN and nPc@MSN. The result shows that the incorporation of cPc and nPc into MSNs can greatly promote the self-quenching not only for fluorescence emission, but also for singlet oxygen production, suggesting that MSNs could serve as a promising platform for the design of self-quenched activatable photosensitizers. 2.0 Absorbance of DPBF at 415 nm

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Figure 3. Comparison of the rate of photodegradation of DPBF sensitized by different ZnPc systems in DMF.

2.3. pH-Responsive Properties. To examine the effect of pH on the fluorescence property of cPc@MSN, the fluorescence spectra of this MSN system in phosphate buffered saline (PBS) at different pH (5.5, 6.0, 6.5, and 7.4) were monitored over 24 h (Figure S2 in Supporting Information). The fluorescence intensity increased gradually with time, and the extent was higher at lower pH. By contrast, for the analogue entrapped with the non11 Environment ACS Paragon Plus


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cleavable dimeric ZnPc (nPc@MSN), both the increase in fluorescence intensity and the effect of pH were negligible (Figure S3 in Supporting Information). Figure 4a shows the percentage of fluorescence recovered for cPc@MSN in PBS at different pH at different time intervals using mPc as reference. It can be seen that nearly 90% of the fluorescence was recovered at pH 5.5, while only less than 10% of the fluorescence was recovered at pH 7.4. For nPc@MSN, no significant difference was observed in the fluorescence recovered at different pH (Figure S4 in Supporting Information). It is believed that the acetal linkers were cleaved under acidic conditions and the ZnPc units in the nanochannels of cPc@MSN were released thereby restoring the fluorescence, but this could not occur for nPc@MSN as the encapsulated dimeric ZnPc units could not be cleaved by acid. To further investigate the pH-responsive photosensitizing property of these MSNs, their singlet oxygen generation efficiency was evaluated after being incubated in PBS at different pH (5.5, 6.0, 6.5, and 7.4) for 24 h. It was found that the singlet oxygen quantum yield, as reflected by the rate of photodegradation of DPBF, increased at lower pH for cPc@MSN (Figure 4b), but for nPc@MSN the rates were similar over the pH range of 5.5-7.4 (Figure S5 in Supporting Information). The singlet oxygen generation efficiency of cPc@MSN was much higher than that of nPc@MSN and became comparable with that of mPc after 24 h at pH 5.5 (Figure S6 in Supporting Information), showing that only cPc@MSN could fully restore its photosensitizing property under these conditions.

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Fluorescence Recovery (%)

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Figure 4. (a) Percentage of fluorescence recovered for cPc@MSN in PBS (with 0.5% Tween 80) at different pH at different time intervals. (b) Comparison of the rate of



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photodegradation of DPBF sensitized by cPc@MSN after being incubated in PBS (with 0.5% Tween 80) at different pH for 24 h.

2.4. In Vitro Studies. The activation of fluorescence of cPc@MSN was also examined in human colon adenocarcinoma HT29 cells using nPc@MSN as the negative control. The cells were incubated with either one of these nanosystems for 12 h, and then the fluorescence and bright field images were captured by a confocal microscope. As shown in Figure 5a, strong intracellular fluorescence could be observed for cPc@MSN, indicating that these nanoparticles were effectively internalized and activated probably inside the acidic subcellular compartments. By contrast, the intracellular fluorescence of nPc@MSN remained very weak, reflecting that the ZnPc units inside the MSNs could not be activated. The intracellular fluorescence intensity of cPc@MSN was about 16-fold higher than that of nPc@MSN (Figure 5b). This “on-off” fluorescence ratio is much higher than that of our previously reported dimeric ZnPc system.19

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(b) 500 Fluorescence Intensity (a.u.)

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nPc@MSN

Figure 5. (a) The intracellular fluorescence (left column) and bright field (right column) images of HT29 cells after incubation with cPc@MSN or nPc@MSN for 12 h. The concentration of the dimeric ZnPc was fixed at 0.5 µM in both cases. (b) Comparison of the intracellular fluorescence intensities of cPc@MSN and nPc@MSN. Data are expressed as the mean ± standard deviation (number of cells = 200). To reveal the subcellular localization property of cPc@MSN, HT29 cells were incubated with this nanosystem and then with LysoTracker, MitoTracker, or ER-Tracker. The cellular images, upon excitation at the phthalocyanine (at 635 nm) and the trackers (at 488 nm), were captured and analyzed. As shown in Figures S8-S10 in Supporting Information, a bright fluorescence was observed in all the cases, but the dye was not localized in these organelles. It seems that after internalization, the nanosystem was activated and the released phthalocyanine moieties were spread in the cytoplasm. The cytotoxicities of cPc@MSN and nPc@MSN against HT29 cells were then examined using MTT assay, which could indirectly reveal the effect of pH on their in vitro photodynamic activity. Figure 6 shows the dose-dependent survival curves for the two MSN systems, both in the absence and presence of light. Both systems showed no cytotoxicity in



the absence of light as many other silica nanoparticles do. However, cPc@MSN became highly potent upon irradiation with an IC50 value of 31 nM (with respect to the concentration of cPc) or 0.56 µg mL-1 (with respect to MSN), while nPc@MSN remained essentially nonphotocytotoxic within the range of concentration tested. The results could again be attributed to the activation of cPc@MSN inside the acidic subcellular organelles. The remarkable difference in photocytotoxicity for the two MSN systems represents a major improvement compared with our previously reported cleavable and non-cleavable dimeric counterparts, which showed similar photocytotoxicity.19 The non-toxic nature of nPc@MSN upon irradiation implies that the cleavable counterpart cPc@MSN would remain non-toxic in physiologically neutral conditions even in the presence of light. This property is essential for photosensitizers that can be selectively activated in the slightly acidic tumor microenvironment while leaving the surrounding healthy tissues unharmed. In addition, the cytotoxicity of cPc@MSN was also compared with that of the free cPc (Figure S7 in Supporting Information). The IC50 value of the latter was found to be 46 nM, which was close to that of cPc@MSN (31 nM). The similar cytotoxicity suggested that the MSN carrier would not affect the photocytotoxicity, and cPc@MSN could be as effective as cPc for PDT. 120 100 % Cell Viability

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Figure 6. Cytotoxic effects of cPc@MSN and nPc@MSN on HT29 in the absence (closed symbols) and presence (open symbols) of light (λ > 610 nm, 40 mW cm-2, 48 J cm-2). Data are expressed as the mean ± standard error of the mean of three independent experiments, each performed in quadruplicate.

2.5. In Vivo Studies. The activation of cPc@MSN was also demonstrated in HT29 xenograft in Balb/c nude mice again using nPc@MSN as the negative control. Figure 7a shows the whole body fluorescence images of the nude mice being treated with an intratumoral dose of cPc@MSN or nPc@MSN (equivalent to 40 nmol cPc or nPc per kg body weight) monitored continuously for 24 h. For the mice being treated with cPc@MSN, the intratumoral fluorescence increased significantly over the first 10 h, while the fluorescence remained very weak in the case of nPc@MSN. The quantified fluorescence intensities in the tumors were also determined and are displayed in Figure 7b. It is clear that cPc@MSN was activated inside the tumor over the first 10 h, leading to a continuous growth of intratumoral fluorescence intensity. The fluorescence intensity at the 24 h time point diminished, but remained visible, suggesting that the released ZnPc units were relatively stable and remained photo-active.

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1500

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Figure 7. (a) Fluorescence images of tumor-bearing nude mice before and after intratumoral injection of cPc@MSN (upper) or nPc@MSN (lower) over 24 h. (b) Corresponding changes in fluorescence intensity per unit area of the tumor. Five mice were used for each MSN system.

3. CONCLUSION A pH-responsive self-quenched ZnPc dimer was conjugated to a MSN platform. The aggregation and subsequently the self-quenching effect of the dimeric ZnPc molecules inside the nanochannels of MSN was greatly promoted, resulting in negligible fluorescence emission and singlet oxygen generation in DMF or PBS. These properties, however, could be greatly restored in acidic media, not only in solution, but also inside tumor cells, with a superior “on-off” ratio. The nontoxic nature of the analogue with non-cleavable dimer (nPc@MSN) upon irradiation suggests that the cleavable counterpart cPc@MSN is a promising candidate that can be selectively activated in the acidic tumor microenvironment, 18 Environment ACS Paragon Plus

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while leaving the surrounding healthy tissues unharmed. The in vivo activation of fluorescence was also demonstrated for cPc@MSN in HT29 xenograft. The overall results suggest that this nanosystem is a smart activatable nanophotosensitizing platform that is potentially useful for advanced PDT.

4. EXPERIMENTAL SECTION General. All the reactions were performed under an atmosphere of nitrogen. DMF and n-pentanol were dried over barium oxide and sodium, respectively, and distilled under reduced pressure. Tetrahydrofuran (THF) was distilled from sodium benzophenone ketyl. Chromatographic purification was performed on silica gel (Macherey-Nagel, 230−400 mesh) with the indicated eluents. Size-exclusion chromatography was carried out on Bio-Beads SX1 beads (200-400 mesh) with THF as the eluent. All other solvents and reagents were of reagent grade and used as received. Compound 1 was prepared as described previously.24 1

H and 13C{1H} NMR spectra were recorded on a Bruker AVANCE III 400 spectrometer

(1H, 400 MHz; 13C, 100.6 MHz) in CDCl3 unless otherwise stated. Spectra were referenced internally by using the residual solvent (for 1H, δ = 7.26) or solvent (for

13

C, δ = 77.16)

resonances relative to SiMe4. Electrospray ionization (ESI) mass spectra were recorded on a Thermo Finnigan MAT 95 XL mass spectrometer. For accurate mass measurements, the lowest m/z value of the isotopic envelope was reported and compared with the theoretical value. TEM images were obtained on a FEI Tecnai G2 Spirit transmission electron microscope operated at 120 keV acceleration voltage. Samples dispersed in Milli-Q water (10 µL) were deposited on carbon film-coated Cu grids (200 mesh) and dried under air before the

TEM analysis. The images were digitized and analyzed using the TEM Imagine and Analysis Software (v421). Dynamic light scattering measurements were performed with a Jianke



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Portable Particle Size Analyzer with a 523 nm laser at room temperature, and the data were correlated by the ALV-Correlator Software. Samples were dispersed in Milli-Q water and sonicated for 15 min before the measurements. Scattering count rate was kept between 3001000 kilocounts per second in each measurement. FT-IR spectra were recorded with a Bruker Alpha FT-IR spectrometer as KBr pellets. UV-Vis and steady-state fluorescence spectra were taken on a Cary 5G UV-Vis-NIR spectrophotometer and a Hitachi F-7000 spectrofluorometer, respectively. The fluorescence quantum yields (ΦF) of the samples (in DMF) were determined by the equation: ΦF(sample) = (Fsample/Fref)(Aref/Asample)(n2sample/n2ref)ΦF(ref),32 where F, A, and n are the measured fluorescence (area under the emission peak), the absorption factor at the excitation position (610 nm), and the refractive index of the solvent, respectively. Unsubstituted ZnPc in DMF was used as the reference [ΦF(ref) = 0.28].33 The singlet oxygen quantum yields (Φ∆) of the samples (in DMF) were determined by the method of

chemical

quenching

of

DPBF

and

the

equation

Φ∆(sample)

=

(Wsample/Wref)(Iref/Isample)Φ∆(ref), where W and I are the DPBF photobleaching rate and the rate of light absorption, respectively. Unsubstituted ZnPc in DMF was used as the reference (Φ∆ = 0.56).34 Preparation of 2. 4-(2-Bromoethoxy)benzaldehyde (750 mg, 3.27 mmol) and 3azidopropan-1-ol

(900

mg,

8.90

mmol)

were

mixed

in

THF

(2

mL).

p-Toluenesulfonic acid (28 mg, 0.16 mmol) was then added and the mixture was stirred at room temperature for 12 h. The reaction was quenched by the addition of Et3N (200 µL) and the volatiles were removed under reduced pressure. The crude product was purified by column chromatography using CHCl3/Et3N (100:1, v/v) as eluent to afford the product as a colorless oil (410 mg, 30%). 1H NMR: δ 7.36 (d, J = 8.8 Hz, 2 H, ArH), 6.91 (d, J = 8.8 Hz, 2 H, ArH), 5.47 (s, 1 H, acetal-H), 4.30 (t, J = 6.4 Hz, 2 H, CH2), 3.58 – 3.66 (m, 4 H, CH2), 3.48 – 3.53 (m, 2 H, CH2), 3.42 (t, J = 6.4 Hz, 4 H, CH2) 1.86 (quintet, J = 6.4 Hz, 4 H, CH2).


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13

C{1H} NMR: δ 158.3, 131.3, 128.0, 114.4, 101.5, 67.9, 62.0, 48.5, 29.2, 29.1. HRMS (ESI):

m/z calcd for C15H21BrN6NaO3 [M+Na]+, 435.0751; found, 435.0755. Preparation of 3. Compound 1 (100 mg, 0.11 mmol), 2 (16 mg, 0.04 mmol), CuSO4⋅5 H2O (10 mg, 0.04 mmol), and sodium ascorbate (16 mg, 0.08 mmol) were dissolved in a mixture of CHCl3, EtOH, and H2O (12:1:1, v/v, 10 mL). The mixture was stirred at room temperature for 24 h, and then the volatiles were removed under reduced pressure. The crude product was first purified by size-exclusion chromatography using THF as eluent, followed by silica gel chromatography using CHCl3/MeOH (20:1, v/v) as eluent. The product was collected as a greenish blue solid (39 mg, 44%). 1H NMR (CDCl3 with a trace amount of pyridine-d5): δ 9.21 (d, J = 7.2 Hz, 4 H, Pc-Hα), 9.15-9.17 (m, 4 H, Pc-Hα), 8.97 (d, J = 7.2 Hz, 4 H, Pc-Hα), 7.97-8.04 (m, 12 H, Pc-Hβ), 7.36 (s, 2 H, triazole-H), 7.12 (s, 4 H, Pc-Hβ), 7.03 (d, J = 8.8 Hz, 2 H, ArH), 6.65 (d, J = 8.8 Hz, 2 H, ArH), 5.02 (s, 1 H, acetal-H), 4.774.81 (m, 8 H, CH2), 4.39-4.42 (m, 12 H, CH2). 4.06-4.14 (m, 14 H, CH2), 3.81-3.86 (m, 8 H, CH2), 3.66-3.72 (m, 8 H, CH2), 3.59-3.61 (m, 4 H, CH2), 3.54-3.57 (m, 4 H, CH2), 3.48-3.52 (m, 2 H, CH2), 3.36 (s, 6 H, CH3), 3.14-3.19 (m, 2 H, CH2), 3.01-3.07 (m, 2 H, CH2), 1.82 (quintet, J = 6.4 Hz, 4 H, CH2). 13C{1H} NMR (CDCl3 with a trace amount of pyridine-d5): δ 158.2, 153.6, 153.5, 153.4, 153.3, 153.2, 152.2, 150.0, 144.9, 138.9, 138.8, 138.5, 138.4, 130.8, 128.8, 127.8, 126.9, 122.7, 122.4, 114.3, 101.6, 72.0, 71.2, 70.9, 70.7, 70.6, 69.8, 68.9, 67.8, 64.5, 61.6, 59.1, 47.1, 30.1, 29.2 (some of the signals were overlapped). HRMS (ESI):

m/z calcd for C111H111BrN22O19Zn2 [M+2H]2+, 1131.3076; found, 1131.3137. Preparation of cPc. A mixture of 3 (50 mg, 22 µmol) and sodium azide (7.3 mg, 112 µmol) in DMF (10 mL) was stirred at 80 oC for 24 h. The solvent was removed at ca. 50 oC

under reduced pressure. The residue was dissolved in CHCl3 and the undissolved solid was filtered off. The filtrate was concentrated under reduced pressure to give the product as a greenish blue solid (49 mg, 99%). 1H NMR (CDCl3 with a trace amount of pyridine-d5): δ



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9.18 (d, J = 7.6 Hz, 4 H, Pc-Hα), 9.12-9.14 (m, 4 H, Pc-Hα), 8.95 (d, J = 7.2 Hz, 4 H, Pc-Hα), 7.93-8.04 (m, 12 H, Pc-Hβ), 7.40 (s, 2 H, triazole-H), 7.11 (s, 4 H, Pc-Hβ), 7.04 (d, J = 8.8 Hz, 2 H, ArH), 6.65 (d, J = 8.8 Hz, 2 H, ArH), 5.05 (s, 1 H, acetal-H), 4.76-4.78 (m, 8 H, CH2), 4.50 (br s, 4 H, CH2), 4.38-4.41 (m, 8 H, CH2), 4.15 (t, J = 6.8 Hz, 4 H, CH2), 4.06-4.10 (m, 8 H, CH2), 3.89 (t, J = 4.8 Hz, 2 H, CH2), 3.80-3.85 (m, 8 H, CH2), 3.66-3.70 (m, 8 H, CH2), 3.61-3.63 (m, 4 H, CH2), 3.50-3.54 (m, 4 H, CH2), 3.41 (t, J = 4.8 Hz, 2 H, CH2), 3.34 (s, 6 H, CH3), 3.17-3.23 (m, 2 H, CH2), 3.04-3.10 (m, 2 H, CH2), 1.85 (quintet, J = 6.0 Hz, 4 H, CH2).

13

C{1H} NMR (CDCl3 with a trace amount of pyridine-d5): δ 158.4, 153.6, 153.5,

153.3, 153.2, 152.2, 152.1, 150.0, 144.9, 138.9, 138.4, 130.8, 128.9, 128.8, 127.8, 126.9, 126.8, 122.6, 122.4, 114.4, 114.3, 114.2, 101.6, 77.4, 72.0, 71.2, 70.9, 70.7, 70.6, 69.9, 68.9, 66.9, 66.7, 64.6, 61.6, 59.1, 50.1, 47.1, 30.1 (some of the signals were overlapped). HRMS (ESI): m/z calcd for C111H110N25NaO19Zn2 [M+H+Na]2+, 1123.8440; found, 1123.8437. Preparation of 4. Cetyl trimethylammonium bromide (CTAB) (0.30 g) was dissolved in aqueous NH3 (0.3 M, 127 mL) to which 1-decane in ethanol (0.6%, v/v, 10 mL) was added. The mixture was sonicated at room temperature for 1 h, and then stirred at 40 oC for 30 min. A solution of tetraethyl orthosilicate (TEOS) in ethanol (0.2 M, 3.0 mL) was added with stirring. The mixture was stirred for 5 h. Another portion of TEOS in ethanol (1.0 M, 3.0 mL) and a solution of (3-aminopropyl)triethoxysilane in ethanol (50 mM, 3.0 mL) were then added simultaneously. Stirring was continued for 1 h and then the mixture was aged at 40 oC for 20 h. The nanoparticles were collected by centrifugation at 14,000 rcf for 40 min, and then washed and redispersed with water (x 3). CTAB was removed by extraction with 0.5 M HCl in ethanol at 70 oC for 12 h twice. The product was collected by centrifugation at 10,000 rcf for 60 min, and then washed and redispersed with ethanol (x 3) and then with water (x 3). Preparation of 5. A mixture of 4 (50 mg), propargyl bromide (80 wt. % in toluene, 84 µL, 0.75 mmol), and K2CO3 (104 mg, 0.75 mmol) in DMF (10 mL) was stirred at room


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temperature for 2 days. 5 was collected by centrifuging at 18,000 rcf for 30 min, and then washed and redispersed with DMF (x 3) and then with water (x 3). Unexpected Synthesis of mPc. mPc was isolated as an unexpected product during the click reaction of cPc (4 mg, 1.8 µmol) with 5 in the presence of CuSO4⋅5 H2O (0.8 mg, 3.2 µmol) and sodium ascorbate (1.3 mg, 6.6 µmol) in a mixture of CHCl3, EtOH, and H2O (12:1:1, v/v, 3 mL). The compound was purified by chromatography on silica gel using CHCl3/MeOH (10:1, v/v) as eluent. 1H NMR (CDCl3 with a trace amount of pyridine-d5): δ 9.31-9.40 (m, 6 H, Pc-Hα), 8.12 (virtual s, 6 H, Pc-Hβ), 7.46 (s, 2 H, Pc-Hβ), 7.39 (s, 1 H, triazole-H), 4.92 (br s, 4 H, CH2), 4.47-4.51 (m, 6 H, CH2), 4.21-4.26 (m, 2 H, CH2), 4.124.13 (m, 4 H, CH2), 3.81-3.86 (m, 4 H, CH2), 3.65-3.70 (m, 4 H, CH2), 3.58 (br s, 2 H, CH2), 3.51-3.53 (m, 2 H, CH2), 3.33-3.35 (m, 5 H, CH2 and CH3), 1.81 (quintet, J = 5.6 Hz, 2 H, CH2).

13

C{1H} NMR (CDCl3 with a trace amount of pyridine-d5): δ 153.9, 153.8, 153.7,

153.6, 153.5, 153.4, 152.4, 150.2, 150.1, 144.8, 144.7, 138.9, 138.5, 129.0, 127.1, 123.1, 122.8, 122.6, 114.7, 72.0, 71.2, 70.9, 70.8, 70.7, 70.6, 69.7, 69.2, 69.1, 64.5, 59.1, 58.3, 58.2, 46.8, 32.5 (some of the signals were overlapped). HRMS (ESI): m/z calcd for C51H52N11O9Zn [M+H]+, 1026.3235; found, 1026.3229. Preparation of cPc@MSN. A mixture of CuI (2 mg, 11 µmol) and Et3N (14 µL, 0.1 mmol) in DMF (2 mL) was prepared. This mixture (20 µL) was then added to a dispersion of 5 (10 mg) and cPc (4 mg, 1.8 µmol) in DMF (2 mL). The resulting mixture was stirred at room temperature for 24 h. The product was collected by centrifuging at 18,000 rcf for 30 min, and then washed and redispersed with sodium N,N-diethyldithiocarbamate (0.1 M in DMF) (x 5) and then with DMF (x 5). Preparation of 7. A mixture of 6 (500 mg, 2.03 mmol) and 10 wt. % Pd/C (11 mg, 10 µmol Pd) in CH2Cl2 (10 mL) was stirred under hydrogen at room temperature for 6 h. The undissolved solid was removed by filtration and the filtrate was removed under reduced



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pressure. The product was collected as a shiny white solid (400 mg, 91%). 1H NMR: δ 6.77 (d, J = 8.8 Hz, 2 H, ArH), 6.64 (d, J = 8.8 Hz, 2 H, ArH), 4.21 (t, J = 6.4 Hz, 2 H, CH2), 3.59 (t, J = 6.4 Hz, 2 H, CH2).

13

C{1H} NMR: δ 151.3, 140.9, 116.6, 116.5, 69.1, 29.6. HRMS

(ESI): m/z calcd for C8H11BrNO [M+H]+, 216.0019; found, 216.0020. Preparation of 8. A mixture of 7 (33 mg, 0.15 mmol) and 3-azido-1-bromopropane (250 mg, 1.52 mmol) in DMF (10 mL) was stirred at 50 oC for 12 h. The solvent was removed at ca. 50 oC under reduced pressure. The crude product was purified by chromatography on silica gel using CH2Cl2 as eluent to afford a colorless oil (30 mg, 51%). 1H NMR: δ 6.85 (d, J = 9.2 Hz, 2 H, ArH), 6.71 (d, J = 9.2 Hz, 2 H, ArH), 4.23 (t, J = 6.4 Hz, 2 H, CH2), 3.61 (t, J = 6.4 Hz, 2 H, CH2), 3.35 (t, J = 6.4 Hz, 4 H, CH2), 3.28 (t, J = 6.4 Hz, 4 H, CH2), 1.80 (quintet, J = 6.4 Hz, 4 H, CH2). 13C{1H} NMR: δ 150.8, 143.1, 116.5, 116.2, 69.0, 49.5, 49.3, 29.6, 26.7. HRMS (ESI): m/z calcd for C14H21BrN7O [M+H]+, 382.0985; found, 382.0993. Preparation of 9. Compound 1 (97 mg, 0.10 mmol), 8 (10 mg, 0.03 mmol), CuSO4⋅5 H2O (0.8 mg, 3.2 µmol), and sodium ascorbate (1.3 mg, 6.6 µmol) were dissolved in a mixture of CHCl3, EtOH, and H2O (12:1:1, v/v, 10 mL). The mixture was stirred at room temperature for 24 h, and then mixed with water (15 mL) followed by extraction with CHCl3 (15 mL × 2). The combined organic layer was dried over anhydrous Na2SO4 and evaporated to dryness under reduced pressure. The crude product was first purified by size-exclusion chromatography using THF as eluent, followed by chromatography on silica gel using CH2Cl2/MeOH (20:1, v/v) as eluent. The product was obtained as a greenish blue solid (25 mg, 43%). 1H NMR (CDCl3 with a trace amount of pyridine-d5): δ 9.21 (d, J = 7.6 Hz, 4 H, Pc-Hα), 9.16-9.18 (m, 4 H, Pc-Hα), 8.95 (d, J = 6.8 Hz, 4 H, Pc-Hα), 7.96-8.03 (m, 12 H, PcHβ), 7.24 (s, 2 H, triazole-H), 7.06 (s, 4 H, Pc-Hβ), 6.41 (d, J = 8.8 Hz, 2 H, ArH), 6.12 (d, J = 8.8 Hz, 2 H, ArH), 4.74-4.78 (m, 8 H, CH2), 4.36-4.40 (m, 8 H, CH2). 4.22 (br s, 4 H, CH2), 4.07-4.11 (m, 8 H, CH2), 3.90 (t, J = 6.0 Hz, 2 H, CH2), 3.83-3.85 (m, 4 H, CH2), 3.79-3.81


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(m, 4 H, CH2), 3.74-3.78 (m, 4 H, CH2), 3.69-3.71 (m, 4 H, CH2), 3.62-3.64 (m, 4 H, CH2), 3.54-3.56 (m, 4 H, CH2), 3.49-3.52 (m, 4 H, CH2), 3.41 (t, J = 6.0 Hz, 2 H, CH2), 3.37 (s, 6 H, CH3), 2.45 (t, J = 6.4 Hz, 4 H, CH2), 1.42 (quintet, J = 6.8 Hz, 4 H, CH2).

13

C{1H} NMR

(CDCl3 with a trace amount of pyridine-d5): δ 153.7, 153.3, 149.9, 144.9, 142.4, 138.9, 138.4, 128.9, 122.8 122.7, 122.4, 118.0, 115.9, 72.1, 71.3, 71.2, 70.9, 70.8, 70.6, 69.9, 68.9, 68.5, 64.4, 59.2, 49.3, 47.6, 29.6, 27.4 (some of the signals were overlapped). HRMS (ESI): m/z calcd for C110H110BrN23O17Zn2 [M+2H]2+, 1097.3557; found, 1097.3539. Preparation of nPc. A mixture of 9 (50 mg, 22 µmol) and sodium azide (7.3 mg, 112 µmol) in DMF (10 mL) was stirred at 80 oC for 24 h. The solvent was removed at ca. 50 oC

under reduced pressure. The residue was dissolved in CHCl3 and the undissolved solid was filtered off. The filtrate was concentrated under reduced pressure to give the product as a greenish blue solid (49 mg, 99%). 1H NMR (CDCl3 with a trace amount of pyridine-d5): δ 9.16–9.22 (m, 8 H, Pc-Hα), 8.90 (d, J = 6.8 Hz, 4 H, Pc-Hα), 7.93–8.06 (m, 12 H, Pc-Hβ), 7.12 (s, 2 H, triazole-H), 6.99 (s, 4 H, Pc-Hβ), 6.37 (d, J = 8.8 Hz, 2 H, ArH), 6.05 (d, J = 8.8 Hz, 2 H, ArH), 4.73-4.74 (m, 8 H, CH2), 4.35-4.38 (m, 8 H, CH2), 4.08-4.10 (m, 8 H, CH2), 3.95 (s, 4 H, CH2), 3.83 (t, J = 5.2 Hz, 4 H, CH2), 3.78 (t, J = 4.8 Hz, 4 H, CH2), 3.69-3.71 (m, 6 H, CH2), 3.65 (t, J = 4.8 Hz, 4 H, CH2), 3.54-3.58 (m, 8 H, CH2), 3.40 (br s, 4 H, CH2), 3.37 (s, 6 H, CH3), 3.33 (t, J = 5.2 Hz, CH2), 2.33 (t, J = 6.4 Hz, 4 H, CH2), 1.33 (quintet, J = 6.4 Hz, 4 H, CH2). 13C{1H} NMR (CDCl3 with a trace amount of pyridine-d5): δ 153.6, 153.4, 153.3, 153.1, 152.1, 151.7, 149.9, 144.9, 142.2, 138.8, 138.4, 138.3, 128.9, 128.8, 126.7, 122.8, 122.7, 122.4, 118.1, 115.4, 114.3, 114.2, 77.4, 72.0, 71.2, 70.9, 70.7, 70.6, 69.8, 68.8, 67.3, 64.5, 59.1, 50.1, 49.3, 47.5, 27.4 (some of the signals were overlapped). HRMS (ESI):

m/z calcd for C110H110N26O17Zn2 [M+2H]2+, 1115.8103; found, 1115.8160. Preparation of nPc@MSN. A mixture of CuI (2 mg, 11 µmol) and Et3N (14 µL, 0.1 mmol) in DMF (2 mL) was prepared. This mixture (20 µL) was then added to a dispersion of



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5 (10 mg) and nPc (5 mg, 2.3 µmol) in DMF (2 mL). The resulting mixture was stirred at room temperature for 24 h. The product was collected by centrifuging at 18,000 rcf for 30 min, and then washed and redispersed with sodium N,N-diethyldithiocarbamate (0.1 M in DMF) (x 5) and then with DMF (x 5). General Procedure for the Ninhydrin Test. The MSNs were dispersed in a ninhydrin solution in ethanol (10 mM, 4 mL). The mixture was stirred at room temperature for 2 h, and then at 70 oC for 15 min. After being cooled to room temperature, it was stirred for a further 1 h. The MSNs were removed by centrifugation at 4,000 rpm for 30 min. The absorption spectrum of the supernatant was recorded. Determination of ZnPc Loading. cPc@MSN (96 µg) was dispersed in DMF (3 mL). The cPc inside the MSNs was cleaved upon addition of HCl (3 M, 1 µL). The Q-band absorbance was measured by UV-Vis spectroscopy and the concentration of ZnPc was quantified by comparing with the Q-band of mPc (λmax = 690 nm, log ε = 5.30), assuming that all cPc was cleaved to mPc. The loading of cPc@MSN was calculated by dividing the concentration of ZnPc by the concentration of MSNs. The loading of ZnPc in nPc@MSN was estimated by comparing its Q-band absorbance with that of cPc@MSN in DMF, assuming that the two dimers have the same aggregation tendency inside the MSNs. Study of pH-Responsive Fluorescence Emission. cPc@MSN and nPc@MSN were first dispersed in DMF respectively (10 mg mL-1 for cPc@MSN and 15 mg mL-1 for nPc@MSN, in which the concentration of cPc and nPc was 0.55 mM). These stock solutions (5.4 µL) were diluted with PBS (3 mL) with 0.5% Tween 80 at different pH (5.5, 6.0, 6.5, and 7.4). The resulting solutions containing 18 µg mL-1 of cPc@MSN or 27 µg mL-1 of nPc@MSN, equivalent to 1 µM of the dimeric ZnPc, and 0.2% (v/v) of DMF were stirred continuously at room temperature. The fluorescence spectra (λex = 610 nm, λem = 630-800 nm) of these solutions were then recorded at different time intervals over a period of 24 h.


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Study of pH-Responsive Singlet Oxygen Generation. The aforementioned PBS solutions of cPc@MSN and nPc@MSN containing 1 µM of the dimeric ZnPc were stirred continuously at room temperature for 24 h. A solution of DPBF in DMF (24 mM, 10 µL) was then added. Before each measurement of the absorbance at 417 nm, the solutions were illuminated with red light from a 200 W halogen lamp after passing through a water tank for cooling and a color glass filter (Newport) cut-on at λ = 610 nm for a particular time interval. The decay of DPBF was monitored for a total irradiation time of 90 s. Cell Line and Culture Conditions. The HT29 human colorectal adenocarcinoma cells (ATCC, no. HTB-38) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Invitrogen, cat. no. 23400-021) supplemented with fetal bovine serum (10%) (Invitrogen, cat. no. 10270-106), and penicillin-streptomycin (100 units mL-1 and 100 µg mL-1, respectively). All the cells were grown at 37 °C in a humidified 5% CO2 atmosphere. Study of Photocytotoxicity. Approximately 2 × 104 HT29 cells per well in the culture medium were inoculated in 96-multiwell plates and incubated overnight at 37 °C in a humidified 5% CO2 atmosphere. Drugs were first dissolved in DMF (equivalent to 0.5 mM cPc or nPc), which were then diluted to respective concentrations (equivalent to 0.50, 0.13, 0.063, 0.031, and 0.016 µM cPc or nPc) with the culture medium. The cells were incubated with 100 µL of these drug solutions for 12 h at 37 °C under 5% CO2. The cells were then rinsed with PBS and refed with 100 µL of the culture medium before being illuminated at ambient temperature. The light source consisted of a 300 W halogen lamp, a water tank for cooling, and a color glass filter (Newport) cut-on at λ = 610 nm. The fluence rate (λ ≥ 610 nm) was 40 mW cm-2. Illumination of 20 min led to a total fluence of 48 J cm-2. Cell viability was determined by means of the colorimetric MTT assay.35 After illumination, the cells were incubated at 37 °C under 5% CO2 overnight. A MTT (Sigma) solution in PBS (3 mg mL-1, 50



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µL) was added to each well followed by incubation for 1.5 h under the same environment. A solution of sodium dodecyl sulfate (Sigma, 10% by weight, 50 µL) was then added to each well. The plate was incubated at 37 °C under 5% CO2 for 20 min, and then 70 µL of isopropyl alcohol was added to each well. Solutions in all wells were mixed until homogenous. Absorbance at 540 nm at each well was taken with a Bio-Rad microplate reader. The average absorbance of the blank wells, which did not contain the cells, was subtracted from the readings of the other wells. The cell viability was then determined by the equation: % viability = [∑(Ai/Acontrol × 100)]/n, where Ai is the absorbance of the ith data (i = 1, 2, ...., n), Acontrol is the average absorbance of the control wells in which the phthalocyanine was absent, and n (= 4) is the number of data points. Confocal Fluorescence Microscopic Studies. Approximately 6 × 105 HT29 cells in RPMI 1640 medium (2 mL) were seeded on a coverslip and incubated overnight at 37 °C with 5% CO2. After removal of the medium, the cells were incubated with cPc@MSN or nPc@MSN (equivalent to 0.5 µM cPc or nPc) for 12 h. The medium was then removed and the cells were rinsed with PBS and viewed with an Olympus FV1000 IX81-SIM confocal microscope equipped a 635 nm diode laser. All the samples were excited at 635 nm and monitored at 650-750 nm. The images were digitized and analyzed using the FV10-ASW software. Subcellular Localization Studies. Approximately 6 × 105 HT29 cells in RPMI 1640 medium (2 mL) were seeded on a coverslip and incubated overnight at 37 °C with 5% CO2. After removing the medium, the cells were incubated with cPc@MSN (equivalent to 2 µM cPc, 2 mL) for 2 h. The medium was then removed and the cells were further incubated with the respective trackers. For the studies using LysoTracker and MitoTracker, the cells were incubated with LysoTracker Green DND-26 (Molecular Probes; 2.0 µM in the culture medium) or MitoTracker Green FM (Invitrogen; 0.25 µM in the culture medium) for 10 min.


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For the study using ER-Tracker, the cells were incubated with ER-Tracker Green (Invitrogen; 0.20 µM in PBS) for 20 min. For all the cases, the cells were then rinsed with PBS and viewed with an Olympus FV1000 IX81-SIM confocal microscope equipped with a 488 nm argon laser and a 635 nm diode laser. All the Trackers were excited at 488 nm and monitored at 500-570 nm. cPc@MSN was excited at 635 nm and monitored at 650-750 nm. The images were digitized and analyzed using the FV10-ASW software. In Vivo Imaging. Female Balb/c nude mice (20-25 g) were obtained from the Laboratory Animal Services Centre at The Chinese University of Hong Kong. All animal experiments had been approved by the Animal Experimentation Ethics Committee of the University. The mice were kept under pathogen-free conditions with free access to food and water. HT29 cells (1 × 107 cells in 200 µL) were inoculated subcutaneously at the back of the mice. Once the tumors had grown to a size of 60-100 mm3, the mice were fed with low fluorescence diet (TestDiet, no. AIN-93M) for 4 days. cPc@MSN or nPc@MSN was first dispersed in distilled water (equivalent to 40 µM cPc or nPc). These solutions (25 µL, equivalent to 1 nmol of cPc or nPc) were injected intratumorally into the tumor-bearing mice. In vivo fluorescence imaging was captured before and after the injection (at different time points) with an Odyssey infrared imaging system (excitation wavelength at 680 nm, emission wavelength at ≥ 700 nm). The images were digitized and analyzed by the Odyssey imaging system software (no. 9201-500). Five mice were used for each MSN system.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.



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Synthesis of mPc and nPc@MSN, ninhydrin test for MSN 4 and 5, FT-IR spectra of 4, 5, cPc@MSN, and nPc@MSN, time-dependent fluorescence spectra of cPc@MSN and nPc@MSN in PBS at different pH, percentage of fluorescence recovered for nPc@MSN in PBS at different pH at different time intervals, comparison of the rate of photodegradation of DPBF sensitized by mPc, cPc@MSN, or nPc@MSN after being incubated in PBS at different pH for 24 h, subcellular localization studies of cPc@MSN, cytotoxic effects of cPc@MSN and cPc on HT29, and 1H and

13

C NMR

spectra of all the new compounds.

AUTHOR INFORMATION Corresponding Authors * For D.K.P.N.: phone, +852-3943-6375; fax, +852-2603-5057; e-mail, [email protected]. * For P.-C.L.: phone, +852-3442-4493; fax, +852-3442-0549; e-mail, [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS P.-C. Lo received a General Research Fund (Project No. 402212) from the Research Grants Council of the Hong Kong Special Administrative Region, China.

ABBREVIATIONS CTAB, cetyl trimethylammonium bromide; DMF, N,N-dimethylformamide; DPBF, 1,3diphenylisobenzofuran; ESI, electrospray ionization; MSN, mesoporous silica nanoparticle; PBS, phosphate buffered saline; PDT, photodynamic therapy; RPMI, Roswell Park Memorial Institute; TEM, transmission electron microscopy; TEOS, tetraethyl orthosilicate; THF,


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tetrahydrofuran; ZnPc, zinc(II) phthalocyanine; ΦF, fluorescence quantum yield; Φ∆, singlet oxygen quantum yield.

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Table of Contents Graphic


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pH-Responsive Dimeric Zinc(II) Phthalocyanine in Mesoporous Silica

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