Nanostructured Phthalocyanine Assemblies with Protein-Driven

Department of Electrical Engineering and Creative IT Engineering, POSTECH, Pohang 37673, Korea. J. Am. Chem. Soc. , 2017, 139 (31), pp 10880–10886. ...
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Nanostructured Phthalocyanine Assemblies with Protein-Driven Switchable Photoactivities for Biophotonic Imaging and Therapy Xingshu Li,†,# C-yoon Kim,‡,# Seunghyun Lee,§,# Dayoung Lee,† Hyung-Min Chung,‡ Gyoungmi Kim,† Si-Hyun Heo,‡ Chulhong Kim,*,§ Ki-Sung Hong,*,‡ and Juyoung Yoon*,† †

Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Korea Department of Medicine, School of Medicine, Konkuk University, Seoul 143-701, Korea § Department of Electrical Engineering and Creative IT Engineering, POSTECH, Pohang 37673, Korea ‡

S Supporting Information *

ABSTRACT: Switchable phototheranostic nanomaterials are of particular interest for specific biosensing, high-quality imaging, and targeted therapy in the field of precision nanomedicine. Here, we develop a “one-for-all” nanomaterial that self-assembles from flexible and versatile phthalocyanine building blocks. The nanostructured phthalocyanine assemblies (NanoPcTBs) display intrinsically unique photothermal and photoacoustic properties. Fluorescence and reactive oxygen species generation can be triggered depending on a targeted, protein-induced, partial disassembly mechanism, which creates opportunities for low-background fluorescence imaging and activatable photodynamic therapy. In vitro evaluations indicate that NanoPcTB has a high selectivity for biotin receptor-positive cancer cells (e.g., A549) compared to biotin receptor-negative cells (e.g., WI38-VA13) and permits a combined photodynamic and photothermal therapeutic effect. Following systemic administration, the NanoPcTBs accumulate in A549 tumors of xenograft-bearing mice, and laser irradiation clearly induces the inhibition of tumor growth.

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effort.10,11 A similar perspective was also reported by Chu et al. in 2015.12 Proteins are vital biomacromolecules that perform various functions within organisms, including energy storage, metabolic reaction catalysis, and cellular function regulation. The abnormal expression of proteins is often related to cancer. Biotin receptors, folic acid receptors, and human carbonic anhydrase II (hCAII) are overexpressed in many kinds of cancer cells.13−17 Therefore, these proteins serve as useful biomarkers for potential cancer diagnosis and tumor-targeting therapy.11,18,19 Currently, several switchable designs, such as peptide molecular beacon,20−23 aptamer,12,24 aggregationinduced emission,25−27 and supramolecular approaches,28−31 have been explored to detect cancer-associated proteins and transport anticancer reagents. The visualized signals and therapeutic activities can be switched on upon the selective recognition of protein biomarkers via affinity labeling, electrostatic interactions, hydrophobic ligand-binding, or specific peptide fragment recognition. These theranostic systems use novel strategies to achieve both monitoring and therapy; however, most of them consist of multiple components to create different functions and even require extra materials to construct and stabilize these systems. These perceived “all-in-

INTRODUCTION

The ability to visualize expressions of biomarkers and deliver bioactive reagents in living organisms is fundamentally important for basic biological studies as well as therapeutic applications.1,2 During the development of treatments for particular diseases, such as cancer therapy, there has been remarkable interest in creating theranostic strategies that possess simultaneous monitoring and therapeutic competencies. With increasing insights into transport processes and biological responses, these theranostic strategies are guiding the medical field toward a new era of more effective and specific treatments.3−5 However, probing low-abundance biomarkers and delivering high loads of therapeutic agents into specific tumor tissues (particularly in combination) remain great challenges. Nowadays, cancer theranostic strategies are typically based on the affinity of antibodies for cancer-associated antigens, which permits the relative accumulation of signal readouts or therapeutic agent payloads in tumor tissues and leads to contrast imaging and targeted treatment.6,7 Unfortunately, the lack of switchable designs, which are used to decrease the required signal intensity and therapeutic agent concentration in tumor tissues relative to normal tissues, causes limited imaging sensitivities with low signal-to-background ratios and increased undesirable side effects.8,9 Thus, the pursuit of switchable designs for cancer theranostics is a main © 2017 American Chemical Society

Received: June 8, 2017 Published: July 14, 2017 10880

DOI: 10.1021/jacs.7b05916 J. Am. Chem. Soc. 2017, 139, 10880−10886

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Journal of the American Chemical Society

longer absorption wavelengths and higher extinction coefficients (typically on the order of 105 M−1·cm−1); thus, Pcs are more suited for optical imaging and phototherapy in biological tissues.39,40 Additionally, several studies have demonstrated that nanostructured Pc assemblies can be produced by controlling the number and length of the hydrophilic linkers (e.g., polyethylene glycol) substituted on Pcs.41−43 Hence, to simplify the structural composition, a flexible and short hydrophilic linker, triethylene glycol (TEG), was selected to be the key moiety of the self-assembling building block (Pc-TEG). Furthermore, the terminal hydroxyl group of Pc-TEG can be easily modified with a small molecular targeting group (e.g., biotin). As shown in Figure 1, candidates containing different

one” systems suffer from certain shortcomings, including multistep fabrication and low reagent loadings. In addition, they require complex toxicity investigations due to the multiple components used and potentially contain heterogeneous formulations that may hamper their future clinical translation.32 Herein, in contrast, we develop a “one-for-all” switchable design based on partial disassembly driven nanotheranostics that are self-assembled using an intrinsically multifunctional building block. In our system, a versatile zinc(II) phthalocyanine (Pc) derivative bearing targeting groups composed of hydrophilic triethylene glycol linkers is designed and synthesized as the building block (Scheme 1). This amphiphilic chemical structure Scheme 1. Schematic Illustration of the Dynamic Pc Assembly and Partial Disassembly Processes as Well as the Switchable Photoactivitya

a

FL: fluorescence. ROS: reactive oxygen species.

facilitates spontaneous assembly to form a stable dispersion with a uniform nanosize distribution in aqueous solutions. Interestingly, these nanostructured Pc assemblies permit partial disassembly in the presence of targeted proteins, which enables switchable photoactivity. As a result, our nanostructured Pc assemblies display highly promising advantages: (1) they consist of only one pure component and self-assemble without required complex processes; (2) the targeted protein-driven fluorescence trigger provides a high potency for cancer imaging with high signal-to-background ratios; (3) the stimuliresponsive reactive oxygen species (ROS) generation highlights the potential application of our assemblies as an activatable photodynamic therapy (PDT) with minimal side effects; (4) the protein-dependent partial disassembly process indicates that the assemblies also possess photothermal and photoacoustic effects to some extent; and (5) this system has a modular and versatile design that enables exchanging different targeting groups for different biomarkers. Below, we introduce how this design can realize the “one-for-all” concept.

Figure 1. Chemical structures of the building blocks, including Pc1TEG, Pc-2TEG, Pc-4TEG, and Pc-4TEG-B. Here, only one of the possible C4h isomers is displayed for the tetra-substituted Pcs.

numbers of TEG were designed: Pc-1TEG, Pc-2TEG, and Pc4TEG. The detailed synthesis procedure for the samples is described in the Supporting Information. All of these building blocks underwent self-assembly to possibly form nanostructures after dissolution in DMSO followed by dilution with water. After comparing their assemblies, we found that Pc-1TEG and Pc-2TEG were not suited for nanoparticle construction due to their serious aggregation (Figure S1). However, Pc-4TEG could form a nanosized dispersion by controlling its concentration (Figure S2). To increase the possible tumor-targeting capability of Pc4TEG, we further linked Pc-4TEG with biotin (Pc-4TEG-B, Figure 1) and studied its self-assembly in water. Interestingly, compared to Pc-4TEG, Pc-4TEG-B could form a more uniform dispersion with a mean hydrodynamic diameter ranging from 100 to 200 nm (Figure S3 and Figure S4). In addition, the Pc4TEG-B assemblies (NanoPcTB) in water were much more stable than the Pc-4TEG assemblies (NanoPcT), especially at high concentrations (Figure S5). We speculate that the more balanced partition ability of Pc-4TEG-B between a hydrophobic solvent and water (Log PO/W = 0.23 ± 0.07, Table S1) made it more suitable to self-assemble into a nanostructure. In addition, the flexible TEG-biotin chains were either stretched into the

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RESULTS AND DISCUSSION Design and Synthesis of Novel One-for-All Building Blocks and Fabrication of Their Nanostructured Assemblies. In recent years, nanostructured porphyrin assemblies (such as the porphyrin-coupled lipid-assembled porphysomes developed by Zheng et al.,33−35 porphyrinphospholipid liposomes developed by Lovell et al.,36,37 and porphyrin-peptide assembled nanodots developed by Yan et al. 38 ) have shown significant potential in theranostic applications. Compared to porphyrins, Pcs generally display 10881

DOI: 10.1021/jacs.7b05916 J. Am. Chem. Soc. 2017, 139, 10880−10886

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concentration of the nanostructured Pc assemblies indicates the concentration of the self-assembled monomers) increased to 67 °C, while the temperature of pure water only increased to 34 °C. This indicated that the NanoPcTBs could convert light energy into heat in an effective and rapid manner, which makes them suited for photothermal therapy (PTT). We also found that the Pc-4TEG-B monomers exhibited minimal photothermal effects (Figure S8), which correlated to their strong fluorescence emission and ROS generation (Table S2). Because the photoacoustic (PA) signal was highly correlated with the photothermal expansion, the NanoPcTB could produce a strong PA signal. Compared with methylene blue (MB), which is a clinically used contrast agent, NanoPcTB produced a much stronger (∼7 times) PA signal (Figure 2f) at 680 nm with the same concentration (200 μM). Switchable Photoactivity Based on Specific ProteinDriven Partial Disassembly. Recently, Hamachi et al. developed several self-assembling triggered-fluorescence nanoprobes for cancer-specific protein detection.49−51 This approach was based on recognition-driven disassembly of the nanoprobes, which induced a clear and triggered fluorescent signal. Inspired by these interesting results, we also explored the protein-responsive properties of the NanoPcTBs. To evaluate their responsive properties in a test tube setting, we initially employed avidin as the targeted protein to simulate biotin receptors, according to previous reports.49,52 As shown in Figure 3a, the fluorescence emission of the NanoPcTBs in water was dramatically increased upon the addition of avidin. However, the increased fluorescence could be subsequently quenched by adding extra biotin. In addition, an increase in fluorescence was not observed when NanoPcT did not contain biotin moieties (Figure 3b). Furthermore, significant changes in the NanoPcTB fluorescence were not observed in the present of various other proteins (Figure 3c), indicating the high selectivity of the assemblies for avidin. These results indicated that the fluorescence turn-on was triggered only by the specific recognition between the biotin ligand and the binding pocket of the avidin; therefore, NanoPcTB is an ideal fluorescent probe for avidin, at least in test tube settings. The ROS generated from NanoPcTB in the presence and absence of avidin were also compared by using 2,7-dichlorofluorescin diacetate as a probe. As shown in Figure 3d and S9, the emission spectra indicated that ROS were not generated by NanoPcTB in the absence of avidin. However, after adding avidin, the ROS generation was clearly recovered. The ROS generation correlated well with the fluorescence emission. Similar results have also been reported in previous photosensitizer-related systems.53,54 This switchable photosensitizing ability suggested that NanoPcTB is highly suited for activatable PDT, which has been demonstrated to be much more promising for cancer treatment than traditional PDT because of its minimal side effects.55−57 As aforementioned, the fluorescence emission and ROS generation abilities of Pcs should influence their photothermal effect and thereby change their PA signal. To directly evaluate the photothermal properties of the Pcs in response to proteins, we compared their thermal behavior before and after adding avidin. From Figure S10, the magnitude of the temperature elevation of NanoPcTB decreased after adding avidin, but the temperature increase was still higher than that of the control group. Similar responses were observed in the PA signal comparison (Figure S11). These results revealed that the NanoPcTBs are applicable as a photothermal and PA contrast

water due to their hydrophilicity or stacked into Pc macrocycles due to hydrogen bonds or metal coordination, reasonably because TEG-biotin is an amphiphilic moiety. As a result, there were more intramolecular and intermolecular interactions in the NanoPcTBs, which resulted in more stabilized nanostructures. In Figure 2a,b, the scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images reveal that

Figure 2. Fabrication of nanostructured Pc-4TEG-B assemblies (NanoPcTB) and their properties. The morphology of NanoPcTB determined using (a) SEM and (b) TEM. The inset presents two pictures of the NanoPcTB dispersion and its Tyndall phenomenon. (c) Electronic absorption and (d) fluorescence spectra (excited at 610 nm, slit 5/5 nm) of the Pc-4TEG-B monomers (1 μM) in DMSO and NanoPcTB (3 μM) in water. (e) Temperature increase of the NanoPcTB in water after irradiation with a 655 nm laser (2.5 W/cm2). (f) Photoacoustic (PA) spectra of the NanoPcTB (200 μM) in water and MB (200 μM) in water.

the NanoPcTBs had nearly regular spherical shapes with a size of approximately 100 nm. Blueshifting and broadening were found for the Soret and Q bands in the absorption spectra of NanoPcTB in water compared to the Pc-4TEG-B monomers in DMSO (Figure 2c), indicating the formation of H-aggregates according to Kasha’s exciton theory,44 revealing the face-to-face molecular stacking in their nanostructures.45−47 The fluorescence spectra indicated that the fluorescence emission of NanoPcTB in water was superquenched (Figure 2d). According to the typical photophysical activation processes of Pcs (Figure S6), the fluorescence emission and intersystem crossing effects were suppressed, indicating the high potential of NanoPcTBs as photothermal agents.48 As shown in Figures 2e and S7, the temperature increase in the assemblies was concentration and light intensity dependent. For example, after 1 min of irradiation with a 655 nm laser at 2.5 W/cm2, the temperature of the NanoPcTBs in water at 12 μM (the 10882

DOI: 10.1021/jacs.7b05916 J. Am. Chem. Soc. 2017, 139, 10880−10886

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the local areas of tumor tissues or cancer cells, we speculate that the NanoPcTBs have potential applications in multimodal photonic imaging and cancer-targeted therapy in vivo. In Vivo Fluorescence Imaging and Photoacoustic Tomography of NanoPcTB. To investigate the applicability of the NanoPcTBs for in vivo fluorescence imaging, they were injected intravenously into mice bearing A549 tumors. At 24 h postinjection, high tumor fluorescence was observed because NanoPcTB accumulated in the tumor and turned on (Figure 4). Recent studies have demonstrated that biotin receptors are

Figure 4. In vivo fluorescence images of mice bearing A549 tumors before and after intravenous injection with NanoPcTB. The experiment was performed in two mice with different tumor locations to show the tumor-targeting ability of NanoPcTB is tumor-location independent.

overexpressed on many cancer cell surfaces, including breast (4T1), cervical (HeLa), lung (A549), ovarian (Ov2008), colon (Colo-26), and renal (RD0995) cancer cell lines.14,58 Therefore, this low-background in vivo fluorescence imaging was potentially caused by an enhanced permeability and retention (EPR) effect and biotin receptor-mediated endocytosis, which induced the partial disassembly of the NanoPcTBs. The possibility of targeting-driven triggered fluorescence was more markedly illustrated when we compared the in/ex vivo fluorescence images of the NanoPcTB- and NanoPcT-injected mice and only observed high tumor fluorescence in the NanoPcTB-injected mice (Figures S13 and S14). In addition, when the tumor tissue was injected with biotin first and then injected with NanoPcTB, its fluorescence intensity was obviously reduced compared to that of the tumor injected with NanoPcTB only (Figure S15). This result further confirmed that NanoPcTB expressed biotin receptor-triggered in vivo fluorescence. As an emerging and noninvasive imaging modality, photoacoustic tomography enables multiscale high-resolution imaging of biological deep tissues using PA effects.59,60 We subsequently examined the unique qualities of NanoPcTB that are inherently suitable for in vivo PA tomography. First, A549 tumor-bearing mice were intravenously injected with NanoPcTB (200 μM, 150 μL), the PA signal was detected using PA tomography and whole-body in vivo PA images of the mice were generated using the data at different time points after injection. The red dotted box (Figure 5a) represents the scanned area. Prior to NanoPcTB injection, we obtained PA maximum-amplitude-projection (MAP) images of the control group (Figure 5a, preinjection image) through lateral (along the y-axis) and vertical (along the z-axis) projections. The control PA MAP images clearly indicate the major blood vessels at 680 nm. The increasing PA signal (Figure 5b), compared to the PA signal in the estimated tumor region of the control image, indicated that the NanoPcTBs accumulated in the tumor tissues via active and passive targeting in a time-dependent

Figure 3. Switchable photoactivity of the NanoPcTBs based on their specific protein-driven partial disassembly. Fluorescence spectra (excited at 610 nm, slit 10/10 nm) of (a) NanoPcTB and (b) NanoPcT (3 μM) in water with and without avidin (3 μM) and biotin (12 μM). (c) Fluorescence intensity (696 nm) responses of NanoPcTB to various proteins in water. 1 Blank; 2 Avidin; 3 BSA; 4 HSA; 5 Protease; 6 Hemoglobin; 7 Lysozyme; 8 Trypsin; 9 IgG; 10 Transferrin; and 11 Fibrinogen. (d) Comparison of the ROS generation of NanoPcTB in water with and without avidin. An ROS probe of 2,7-dichlorofluorescin diacetate in water was used as the control group. (e) NanoPcTB morphology changes in water before and after adding avidin and after standing for 24 h, as determined using TEM. The right column illustrates the proposed mechanisms for the morphology changes. Avidin/NanoPcTB mole ratio is 1/1.

agent, and they even recover their fluorescence emission and ROS generation upon stimulation by avidin. Subsequently, the probable mechanism behind this avidinresponsive process was investigated. Compared to the photoactivity of the Pc-4TEG-B monomers in DMSO (or DMF), the fluorescence emission of the NanoPcTBs with an equimolar amount of avidin was only partially recovered, and their photothermal effect was partially reduced. In addition, the recovered fluorescence intensity of NanoPcTB was not proportional to the increasing avidin/NanoPcTB mole ratio (Figure S12). Even under a high mole ratio of 10/1 (avidin/ NanoPcTB), the recovered fluorescence intensity of NanoPcTB was still lower than that of the Pc-4TEG-B monomers. Therefore, we speculated that a few Pc-4TEG-B units on the surface of the nanoparticles could be pulled off due to the strong binding affinity between the biotin moieties and avidin. However, the inner biotin moieties were hidden and formed stronger intramolecular and intermolecular interactions, which stabilized and prevented the inner nanostructure from completely disassembling. This partial disassembly process was confirmed from the TEM images (Figure 3e). Given the overexpressed but limited concentrations of biotin receptors in 10883

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NanoPcT under dark conditions. However, under 655 nm laser irradiation, the cell killing effects caused by NanoPcTB and NanoPcT differed greatly. The HeLa cell inhibition induced by NanoPcTB at a concentration of 3 μM under 2.5 W/cm2 laser irradiation was approximately 54%, which was more than 2-fold higher than that of NanoPcT (approximately 21% cell inhibition). In addition, even under a low laser power irradiation (0.22 W/cm2), NanoPcTB still induced approximately 30% HeLa cell death, while NanoPcT exhibited a negligible effect. To illustrate the different cellular damage mechanisms of the two samples, the cellular temperature change and ROS generation after treatment were evaluated. As shown in Figure 6b,c, NanoPcTB and NanoPcT exhibited similar heating effects, while only NanoPcTB induced obvious ROS generation in the biotin receptor-positive cells (A549 and HeLa). These results indicated that NanoPcTB possesses combined PDT and PTT effects, which are enhanced by the partial disassembly induced by the overexpressed biotin receptors on A549 and HeLa cells. Additionally, the much lower ROS generation induced by NanoPcTB in the biotin receptor-negative cells (WI38-VA13) further confirmed the cancer cell-specific phototherapeutic effect of NanoPcTB. To demonstrate the in vivo photonic therapeutic potential of NanoPcTB, we carried out preliminary experiments using NanoPcTB for cancer phototherapy. Four groups of A549 tumor-bearing mice (8 mice per group) were treated with saline, saline combined with laser irradiation, NanoPcTB, and NanoPcTB combined with laser irradiation. The results (Figure 7a,b) show that approximately 40% of the tumor growth was inhibited after 16 d in mice treated with NanoPcTB and then

Figure 5. In vivo photoacoustic tomography of mice bearing A549 tumors before and after intravenous injection of NanoPcTB. (a) Picture of the mouse and relative side-view and top-view 2D-MAP images at 680 nm. (b) Number of pixels in the tumor region that are greater than the constant PA intensity (0.5) as a function of time.

manner. Thus, based on their unique self-quenching and nanostructured properties, the NanoPcTBs are intrinsically suited for photoacoustic tomography and targeting-triggered fluorescence imaging with high signal-to-background ratios. Cancer Therapeutic Efficacy of NanoPcTB. For the therapeutic efficacy evaluation, we first studied the in vitro anticancer effect of NanoPcTB in A549 cells (biotin receptor positive), HeLa cells (biotin receptor positive), and WI38VA13 cells (biotin receptor negative). As shown in Figure 6a, significant toxicity was not observed for both NanoPcTB and

Figure 7. In vivo anticancer effect. (a) Tumor growth curves in mice with A549 xenografts after different treatments: saline intravenous injection without laser irradiation (black curve), saline intravenous injection with laser irradiation (green curve), NanoPcTB intravenous injection without laser irradiation (blue curve), and NanoPcTB intravenous injection with laser irradiation (red curve). Drug dose: 200 μL, 60 μM. Laser conditions: 655 nm, 2.0 W/cm2, and 1 min and then 655 nm, 0.22 W/cm2, and 5 min. Drug light interval: 24 h. * P < 0.05. (b) Representative photograph of the tumors at the end of the in vivo anticancer studies. (c) H&E staining and TUNEL assay results of the tumor sections from the mice after treatment.

Figure 6. In vitro anticancer effect. (a) Cytotoxic effects and (b) temperature of the HeLa cells incubated with NanoPcT and NanoPcTB (3 or 6 μM, respectively) for 2 h without or with 655 nm laser irradiation (0.22 or 2.5 W/cm2, respectively, 5 min). The data are expressed as the mean ± standard deviation (n = 3). (c) ROS generation induced by NanoPcT and NanoPcTB (3 μM) in the A549, HeLa, and WI38-VA13 cells in the presence of laser irradiation (655 nm, 0.22 W/cm2, 5 min). Scale bar = 20 μm. 10884

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irradiated. In contrast, mice treated with NanoPcTB without irradiation showed a significant level of tumor growth, which was comparable to the growth in the mice treated with saline both with and without irradiation. To further confirm the in vivo phototherapeutic efficacy, after these treatments, tumor sections were examined using hematoxylin and eosin (H&E) staining and a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay. The H&E-stained tumors treated with NanoPcTB combined with laser irradiation showed characteristic apoptotic cells, including darkly stained and condensed chromatin, which were not observed in the other groups of tumors (Figure 7c, H&E staining). Moreover, the TUNEL assay results revealed a significant increase in the amount of apoptotic chromatin in the tumors treated with NanoPcTB combined with laser irradiation (Figures 7c and S16). In contrast, negligible necrotic or apoptotic tumor cells were observed in the tumor sections treated only with NanoPcTB or laser irradiation. Despite these encouraging results, we believe that a higher in vivo phototherapeutic efficacy could be obtained after conducting a more systemic evaluation including the optimization of the drug does and light conditions.

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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/jacs.7b05916. Detailed materials and methods, synthesis, characterization, and supplementary figures (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*[email protected] *[email protected] *[email protected] ORCID

Juyoung Yoon: 0000-0002-1728-3970 Author Contributions #

X.L., C.K., and S.L. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS J.Y. is thankful for the support from the National Research Foundation of Korea (NRF), which was funded by the Korea government (MSIP) (No. 2012R1A3A2048814). K.S.H. is thankful for the support from the Ministry for Health and Welfare (HI14C3365) and the Ministry of Education (NRF432 2016R1D1A3B03932998). C.K. is thankful for the support from ICT Consilience Creative Program (IITP-R0346-161007) and Korea Health Technology R Project (HI15C1817).

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CONCLUSION Because of their pure, single-component and multifunctional optical properties, the nanostructured NanoPcTBs are distinguished from previously described porphyrin assemblies and BODIPY assemblies. Although Yan’s porphyrin-peptide assembled nanodots (PPP-NDs) consist of single components, they only possess intrinsically photothermal and photoacoustic properties and are unable to perform biomarker detection.38 Hamachi’s BODIPY assemblies enable target-induced triggered fluorescent signals, which are suited for protein biomarker detection, but they cannot act as therapeutic agents.50 Zheng and Lovell’s porphyrin-lipid assemblies enable multimodal biophotonic imaging and therapy, but they still require extra adjuvants, such as polyethylene glycol and cholesterol, to formulate the nanostructures.33,36 Although the performance of the NanoPcTBs in this article clearly demonstrates their superior characteristics, further investigations are required to gain insight into the mechanisms of their dynamic assemblies and action in vivo. In summary, for the first time, we developed nanostructured Pc assemblies using self-assembled single Pc-4TEG-B monomers as multifunctional phototheranostic agents. Because of the unique noncovalent interactions of the targeted groups and Pcs, these spherical nanoassemblies exhibit targeted protein-driven partial disassembly. As a result, NanoPcTBs are effective agents for cancer-specific triggered-fluorescence imaging and activatable PDT. Additionally, NanoPcTBs exhibit nanoscale optical properties and are intrinsically suited for multimodal imaging and therapeutic applications. Different small molecular targeting groups, such as folic acid and arylsulfonamide ligands, could be easily adopted to potentially direct nanostructured Pc assemblies toward a range of different target biomarkers. Therefore, nanostructured Pc assemblies formed due to the self-assembly of one-for-all building blocks show flexibility and versatility for cancer diagnosis and therapy. This study provides a novel perspective for designing supramolecular phototheranostic agents that may advance toward clinical translation and act as new nanomedicines.

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DOI: 10.1021/jacs.7b05916 J. Am. Chem. Soc. 2017, 139, 10880−10886