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Smart Peptide-Based Supramolecular Photodynamic MetalloNanodrugs Designed by Multicomponent Coordination Self-Assembly Shukun Li, Qianli Zou, Yongxin Li, Chengqian Yuan, Ruirui Xing, and Xuehai Yan J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 13 Aug 2018 Downloaded from http://pubs.acs.org on August 13, 2018

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Journal of the American Chemical Society

Smart Peptide-Based Supramolecular Photodynamic MetalloNanodrugs Designed by Multicomponent Coordination SelfAssembly Shukun Li,†,§,# Qianli Zou,†,# Yongxin Li,†,§ Chengqian Yuan,† Ruirui Xing,† and Xuehai Yan*,†,‡,§ †

State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China

‡

Center for Mesoscience, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China

§

University of Chinese Academy of Sciences, Beijing 100049, China KEYWORDS: metallo-nanodrugs, self-assembly, peptides, photosensitizers, photodynamic therapy ABSTRACT: Supramolecular photosensitizer nanodrugs that combine the flexibility of supramolecular self-assembly and the advantage of spatiotemporal, controlled drug delivery are promising for dedicated, precise non-invasive tumor therapy. However, integrating robust blood circulation and targeted burst release in a single photosensitizer nanodrug platform that can simultaneously improve the therapeutic performance and reduce side effects is challenging. Herein, we demonstrate a multicomponent coordination self-assembly strategy that is versatile and potent for the development of photodynamic nanodrugs. Inspired by the multicomponent self-organization of polypeptides, pigments, and metal ions in metalloproteins, smart metallo-nanodrugs are constructed based on the combination and cooperation of multiple coordination, hydrophobic and electrostatic non-covalent interactions among short peptides, photosensitizers and metal ions. The resulting metallo-nanodrugs have uniform sizes, welldefined nanosphere structures, and high loading capacities. Most importantly, multicomponent assembled nanodrugs have robust colloidal stability and ultrasensitive responses to pH and redox stimuli. These properties prolong blood circulation, increase tumor accumulation, and enhance the photodynamic tumor therapeutic efficacy. This study offers a new strategy to harness robust, smart metallo-nanodrugs with integrated flexibility and multifunction to enhance tumor-specific delivery and therapeutic effects, highlighting opportunities to develop next-generation, smart photosensitizing nanomedicines.

Introduction Photodynamic therapy (PDT) is an emerging therapeutic modality based on combining photosensitive drugs (photosensitizers) and light with endogenous oxygen to collectively exert a selective cytotoxicity towards neoplastic and nonmalignant diseases through complex cascades of photochemical and biological reactions.1-3 With advances in nanotechnology, nanoengineered photosensitizers have been proposed to improve the precision and efficiency of PDT for tumor eradication by enhancing the selective spatiotemporal distribution of photosensitizers while decreasing the likelihood of off-target side effects.4-7 Since photosensitizer aggregation in nanodrugs inhibits the generation of reactive oxygen species (ROS), a burst release of photosensitizers once the nanodrugs accumulate in the targeted cells is desired.8,9 Hence, a variety of nanodrugs based on liposomes, and polymeric and inorganic nanomaterials have been investigated to tailor pharmacokinetics and therapeutic performances of photosensitizers.10-12 Nonetheless, these photosensitizer nanodrugs have several disadvantages. First and foremost, simultaneous integration of robust blood circulation and a targeted burst release in single photosensitizer nanodrugs has not yet been achieved;13,14 Second, the encapsulation of photosensitizers by drug delivery vehicles readily leads to a low

drug-loading capacity and a high premature drug release;12 Third, self-assembled nanodrugs based on non-covalent interactions are prone to quick disassembly upon dilution, diminishing the size-dependent merits of nanodrugs;15 Finally, the synthetic components used in fabricating nanodrugs may cause adverse effects associated with toxic, immune, and inflammatory responses.16 Notwithstanding these defects, the rational engineering of desirable photosensitizer nanodrugs is currently vigorously pursued. However, one strategy or methodology often overlooked is the biological self-assembly involving multiple, reciprocal biomolecules and interactions. A large number of proteins in natural organisms are metalloproteins, which integrate both metal ions and organic cofactors through coordination interactions.17-19 The cooperative coordination of histidine residues and porphyrin derivatives occurs in a variety of biologically important metalloproteins such as haemoglobin.20 In light of this, herein we report a novel multicomponent coordination self-assembly strategy based on combination of histidine-containing short peptides, photosensitizers, and metal ions to design and engineer metallo-nanodrugs for antitumor therapy (Figure 1). Short peptides have shown flexibility and versatility in designing self-assembled materials for biomedical applications due to unique advantages such as programmable primary

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Figure 1. Schematic illustration of supramolecular metallo-nanodrugs for efficient antitumor PDT. (a) Biological cooperative coordination of the heme group in human hemoglobin to histidine through a metal ion (PDB: 1A3N). (b) Construction of metallo-nanodrugs through cooperative coordination of small peptides and photosensitizers in the presence of zinc ions. (c) Schematic molecular organization pattern of a metal-binding peptide and Zn2+. (d) Accumulation of metallo-nanodrugs in tumors during robust blood circulation. (e) Cellular internalization of metallo-nanodrugs through endocytosis, burst release of metallo-nanodrugs in response to cellular compartment environments, and activation of the released photosensitizer to generate toxic ROS for efficient PDT.

sequence, tunable morphology, good biocompatibility and low immunogenicity as well as easy availability.21-24 Therefore, we purposely designed histidine-containing dipeptide or amphiphilic histidine derivative as a starting building block for the coordination self-assembly. Spherical metallo-nanodrugs are readily obtained by zinc ion-coordinated multicomponent self-assembly of the designed histidine-containing peptide or

histidine derivative and a photosensitizer. Intriguingly, metallo-nanodrugs show robust colloidal stability under normal biological conditions and burst release behaviour in tumor microenvironments, e.g., lower pH values and higher glutathione (GSH) levels. These behaviours are based on the robustness and flexibility of multiple coordination interactions with short peptides and photosensitizers. The assembled


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Figure 2. Morphology, coordination pattern, FTIR spectra, and ultrasensitive response of Fmoc-H/Zn2+ and Z-HF/Zn2+ nanoparticles. (a) DLS profiles with pictures of the samples shown in the inset. (b) SEM images. (c) TEM images. (d) Proposed spatial coordination conformation of the peptide and Zn2+ in Fmoc-H/Zn2+ nanoparticles and the lengths of the coordination bonds. (e) Experimental and calculated FTIR spectra. (f, g) Schematic illustration and pictures showing the ultrasensitive response of Fmoc-H/Zn2+ to pH and GSH changes.

metallo-nanodrugs have prolonged blood circulation, improved tumor accumulation, and enhanced tumor ablation. Furthermore, metallo-nanodrugs show negligible in vitro and in vivo toxicities. In contrast to individual photosensitizers and conventional drug-delivery platforms, the metallo-nanodrugs integrate robust blood circulation and targeted burst release in a single nanodrug for the first time. This study provides a promising strategy to develop next-generation smart nanomedicines for precision therapy. Results and discussion Histidine is an amino acid that has a strong coordination ability with metal ions.25 Hence, two histidine-containing compounds, fluorenylmethoxycarbonyl-L-histidine (Fmoc-H), an amphiphilic amino acid, and N-benzyloxycarbonyl-Lhistidine-L-phenylalanine (Z-HF), an amphiphilic short peptide, were chosen as the metal-binding building blocks for the coordination self-assembly. We first investigated coordination self-assembly of Fmoc-H and Z-HF with metal ions. Upon the addition of a solution of zinc chloride (ZnCl2) to a solution of Fmoc-H or Z-HF, an opaline, turbid, colloidal suspension was obtained (Figure 2a), which indicated the formation of nanoparticles. Many essential metal elements

have been widely investigated for biomedical applications due to their unique biological functions and good biocompatibility.26 Hence, Zn2+, a typical essential metal element, was selected for the coordination self-assembly. The dynamic light scattering (DLS) profiles of the resulting suspensions show that the Fmoc-H/Zn2+ and Z-HF/Zn2+ nanoparticles have narrow size distributions and average diameters of 75 ± 16 nm and 78 ± 21 nm, respectively. These size distributions are in the size range suitable for an enhanced permeability and retention (EPR) effect.27 The DLS results also revealed that the two kinds of nanoparticles have almost the same zeta potential values, i.e., -20 ± 1.3 mV for FmocH/Zn2+ and -20 ± 2.5 mV for Z-HF/Zn2+. The scanning electron microscopy (SEM) images show that both FmocH/Zn2+ and Z-HF/Zn2+ are spherical nanoparticles with sizes of approximately 70 nm, which is consistent with the DLS results (Figure 2b). The transmission electron microscopy (TEM) images further confirm that Fmoc-H/Zn2+ and ZHF/Zn2+ are spherical, solid nanoparticles with uniform size distributions (Figure 2c). Subsequently, the self-assembly mechanism of the nanoparticle formation was investigated by quantitative stoichiometry analysis and Fourier transform infrared (FTIR)



spectroscopy. The quantitative component analysis with UVvis spectroscopy and atomic absorption spectroscopy revealed that the molar ratios of Fmoc-H or Z-HF to Zn2+ are close to 2:1 (Table S1 in the Supporting Information). Based on this stoichiometry, the molecular spatial coordination conformation of Fmoc-H or Z-HF with Zn2+ was simulated by a density functional theory (DFT) method. The results showed that the zinc coordination spheres in Fmoc-H/Zn2+ and Z-HF/Zn2+ have four peptide molecules,28 and each peptide molecule provides an imidazole group or a carboxyl group for coordination (Figure 2d and S1). The FTIR spectra confirmed that both the imidazole and carboxyl groups of Fmoc-H or Z-HF coordinate with the Zn2+ in the nanoparticles (Figure 2e and Table S2). The FTIR spectrum of FmocH/Zn2+ has three characteristic bands. The two bands identified at 1500 and 3277 cm-1 are due to the imidazole groups, and the band at 1608 cm-1 is due to the carboxyl group.29,30 Compared to the corresponding bands in the FTIR spectrum of Fmoc-H, all three bands significantly shifted to lower wavenumbers, indicating the imidazole and carboxyl groups coordinated to Zn2+. In addition, the experimental FTIR spectra of Fmoc-H/Zn2+ were consistent with the simulated spectra for the proposed complexes of Fmoc-H and Zn2+, which further confirmed the coordination pattern between Fmoc-H and Zn2+. The same coordination pattern was also suggested when comparing the FTIR spectra of ZHF/Zn2+ and Z-HF. The stoichiometry and FTIR spectra indicate that the formation of Fmoc-H/Zn2+ and Z-HF/Zn2+ involves the primary formation of coordination complexes and further stacking of the resulting complexes. The stacking is the result of multiple, additional non-covalent interactions, such as hydrophobic interactions and π-π stacking of the aromatic motifs.31,32 Not surprisingly, other metal ions, such as Cu2+, can also interact with the peptide building blocks and induce the formation of well-defined nanoparticles (Figure S2). Because the formation of Fmoc-H/Zn2+ and Z-HF/Zn2+ is based on the synergy of the coordination and other noncovalent interactions, the complexes are susceptible to environmental variations. This susceptibility can be exploited to design stimuli-responsive nanoparticle disassembly.33-35 The turbidity of the Fmoc-H/Zn2+ and Z-HF/Zn2+ solutions immediately vanished upon decreasing the pH below 5.0 or increasing the pH above 8.5, which indicated an ultrasensitive pH responsiveness (Figure 2f). The response to the pH decrease can be attributed to the protonation of the imidazole groups at a pH below 5.0, weakening the coordination between histidine and Zn2+. The response to the pH increase can be attributed to the improved solubility of Fmoc-H or Z-HF at a pH above 8.5, which diminished the self-assembly hydrophobic interactions. In addition, Fmoc-H/Zn2+ and ZHF/Zn2+ are also responsive to GSH, which can competitively coordinate with Zn2+ and induce an immediate disassembly of the nanoparticles (Figure 2g). Responsiveness of FmocH/Zn2+ and Z-HF/Zn2+ at pH 4.0 and 9.0, and in the presence of 3 mM GSH was investigated by DLS and TEM. The measurements did not show discernable assemblies (data not shown), indicating the complete disassembly of nanoparticles in response to these changes. Decreased pH values and elevated GSH levels are found in tumor tissues,36-38 especially in intracellular compartments.39-41 Hence, the ultrasensitive

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responsiveness of Fmoc-H/Zn2+ and Z-HF/Zn2+ to pH and GSH variations highlights their potential as smart nanodrug platforms for antitumor therapy. Based on the robust coordination self-assembly of Fmoc-H and Z-HF with Zn2+, we next investigated the feasibility for construction of metallo-nanodrugs with consideration of a drug component inclusion. To this end, chlorin e6 (Ce6), a photosensitizer containing one metal-binding chlorin ring and three carboxyl groups,42 was selected as a drug model. Multicomponent, cooperative coordination of Ce6 and FmocH or Z-HF in the presence of Zn2+ via a one-step self-assembly strategy generates dark colored colloidal suspensions with average sizes of 79 ± 21 nm and 76 ± 21 nm for FmocH/Zn2+/Ce6 and Z-HF/Zn2+/Ce6, respectively (Figure 3a). The SEM and TEM images reveal that both FmocH/Zn2+/Ce6 and Z-HF/Zn2+/Ce6 are spherical, solid nanoparticles, and their size distributions and morphologies show no significant changes compared to those of FmocH/Zn2+ and Z-HF/Zn2+ (Figure 3b and c), respectively. Significantly, the Ce6 loading capacities of Fmoc-H/Zn2+/Ce6 and Z-HF/Zn2+/Ce6 were higher than 50%, and the encapsulation efficiencies exceeded 99% (Table S3). Such loading capacities are much higher than those achieved by carrier-based drug-delivery platforms (typically < 20%)12 and indicate that Ce6 not only acts as a cargo but also participates in the formation of the metallo-nanodrugs through cooperative coordination with the peptide to Zn2+. This participation ensures a high loading capacity. The coordination of Ce6 to Zn2+ was also evident in the UV-vis absorption spectra of Fmoc-H/Zn2+/Ce6 and Z-HF/Zn2+/Ce6 (Figure 3d). Compared to the spectrum of monomeric Ce6, FmocH/Zn2+/Ce6 showed a redshifted Soret band at 416 nm and a blueshifted Q band at 640 nm, which suggested the insertion of Zn2+ into the chlorin ring of Ce6. Additionally, compared to the spectrum of the Ce6/Zn2+ aggregates, the Soret band of Fmoc-H/Zn2+/Ce6 redshifted by 4 nm, and the Q band of Fmoc-H/Zn2+/Ce6 redshifted by 8 nm, which indicated that the Ce6/Zn2+ complex interacts with the peptide building blocks by binding to the histidine group along with other noncovalent interactions,43,44 such as hydrophobic interactions and π-π stacking between the Fmoc groups and the chlorin rings. A similar interaction pattern was also observed when comparing the spectra of Z-HF/Zn2+/Ce6, monomeric Ce6, and Ce6/Zn2+ aggregates. These results confirmed that cooperative coordination of Ce6 and Fmoc-H or Z-HF occurs in the formation of the Fmoc-H/Zn2+/Ce6 and Z-HF/Zn2+/Ce6 metallo-nanodrugs, and Ce6 molecules exist in an aggregated form in the metallo-nanodrugs. For in vivo drug delivery, nanodrug stability during blood circulation is highly important and has been a formidable challenge for self-assembled nanodrugs due to the fragility of non-covalent interactions.15,45,46 Compared to common noncovalent interactions, metal coordination interactions are strong due to their nearly covalent characteristics, and the incorporation of coordination interactions is a possible method to engineer stable, self-assembled metallo-nanodrugs. The stability of Fmoc-H/Zn2+/Ce6 and Z-HF/Zn2+/Ce6 was first investigated by creating 10-fold (v/v) dilutions of their suspensions with phosphate buffered saline (PBS) (pH 7.4) and incubating the suspensions at 37 °C for 24 h. The results


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Figure 3. Morphology, UV-vis absorption spectra, stability, and response of the metallo-nanodrugs. (a) DLS profiles with the pictures of the samples shown in the inset. (b) SEM images. (c) TEM images. (d) UV-vis absorption spectra of Fmoc-H/Zn2+/Ce6, Z-HF/Zn2+/Ce6, monomeric Ce6, and Ce6/Zn2+ aggregates. (e) Size and polymer dispersity index (PDI) of diluted metallo-nanodrugs during incubation at 37 °C for 24 h. (f) Size and PDI of metallo-nanodrugs during incubation in a PBS buffer (pH 7.4) supplemented with 10% (v/v) FBS at 37 °C for 24 h. (g) Ce6 release profiles from Fmoc-H/Zn2+/Ce6 in different release buffers. Unencapsulated Ce6 was used as a control group. The lines are the fitted results according to the Gompertz kinetic release model. Error bars denote the standard deviation (n = 3). *P < 0.05 (one-way ANOVA).

show that both Fmoc-H/Zn2+/Ce6 and Z-HF/Zn2+/Ce6 are stable under such conditions since their average sizes and size distributions were not altered by the treatment (Figure 3e). Then, the stability of the metallo-nanodrugs was investigated by incubating them in PBS (pH 7.4) supplemented with 10% (v/v) fetal bovine serum at 37 °C for 24 h. No discernible changes were observed in the average sizes and size distributions of Fmoc-H/Zn2+/Ce6 and Z-HF/Zn2+/Ce6 (Figure 3f), showing that the metallo-nanodrugs engineered by a combination of cooperative coordination and noncovalent interactions are highly stable under normal physiological conditions. In an aggregated state, the ROS generation capability of photosensitizers is inhibited due to self-quenching, which decreases their PDT efficacy. Hence, the burst release of photosensitizers as monomers from nanodrugs once they reach tumor or intracellular environments is important for efficient PDT.47 The release of Ce6 from the metallo-nanodrugs was evaluated by monitoring the release profiles in various buffer solutions (Figure 3g). The profiles followed the Gompertz model for drug release kinetics.48 The release efficiency of Ce6 from FmocH/Zn2+/Ce6 at pH 7.4 was low, and approximately 18% of the loaded Ce6 was released in 12 h. Beyond 12 h, the release efficiency slowly increased to 35% in 48 h. This low release

efficiency under a pH of 7.4 confirmed the stability of the metallo-nanodrugs and is favorable for improving their blood circulation. When the release buffer pH decreased to 6.5 or 3 mM GSH was added, the release rate significantly accelerated. The release efficiency reached 42% in 12 h at pH 6.5 in the presence of 3 mM GSH and further increased to 80% in 48 h. This release profile is close to that of unencapsulated Ce6, indicating that a burst release of Ce6 from the metallonanodrugs can be readily achieved by varying the pH and GSH levels. Compared to a biologically neutral pH of 7.4, tumor tissues show an enhanced acidity in the interstitial space (pH 6.5-7.2), intracellular endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0).36,39 In addition, tumor tissues also show an approximately four-fold increased GSH level compared to that of normal tissues, and the GSH level in the cytosol (approximately 5 mM) is significantly higher than that in the plasma (approximately 10 μM).37,40,49 Hence, the ultrasensitive responsiveness of the metallo-nanodrugs to pH and GSH levels along with their high stability under normal physiological conditions imply they have a robust blood circulation ability and targeted Ce6 burst release once the metallo-nanodrugs accumulate in tumor tissues or are internalized by tumor cells.



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Figure 4. In vitro PDT evaluation of Fmoc-H/Zn2+/Ce6 and Z-HF/Zn2+/Ce6 on MCF-7 cells. (a) CLSM images of cells incubated with the metallo-nanodrugs (equivalent concentration of Ce6: 10.0 μg mL-1) showing enhanced intracellular uptake and release of the metallo-nanodrugs under prolonged incubation times. (b) A CLSM fluorescence image showing the intracellular location of FmocH/Zn2+/Ce6. The cells were incubated with Fmoc-H/Zn2+/Ce6 (concentration of Ce6: 10.0 μg mL-1) for 24 h and stained with Hoechst 33342 and Alexa 488 stains for the nuclei and membranes, respectively. The red signal is indicative of released Ce6. (c) Flow cytometry analysed fluorescence intensity of cells incubated with the metallo-nanodrugs or unencapsulated Ce6 (equivalent concentration of Ce6: 2.5 μg mL-1). (d) Fluorescence intensity of cells showing the ROS generated by the combination of irradiation and the metallo-

nanodrugs. The cells were incubated with the metallo-nanodrugs or unencapsulated Ce6 (equivalent concentration of Ce6: 2.5 μg mL-1) for 24 h and 10 µM DCFH-DA for 0.5 h followed by the irradiation through a 635 nm laser (3 mW) for 2 min. (e) CLSM images showing the selective inhibition of cells by PDT using the metallo-nanodrugs. The cells were incubated with Fmoc-H/Zn2+/Ce6 (concentration of Ce6: 10.0 μg mL-1) for 24 h, and selected cells were irradiated by a 635 nm laser for 10 min (3 mW). After irradiation, the cells were stained with Hoechst 33342 and PI. The green signal from PI is indicative of dead cells. (f) Viability of the cells treated with Fmoc-H/Zn2+/Ce6, Z-HF/Zn2+/Ce6, or unencapsulated Ce6 in the presence or absence of light irradiation. Scale bars denote 20 μm. Error bars denote the standard deviation (n = 3). *P < 0.05 (one-way ANOVA).

Encouraged by their desirable stability and smart responsiveness, Fmoc-H/Zn2+/Ce6 and Z-HF/Zn2+/Ce6 were further investigated for in vitro PDT. Confocal laser scanning microscopy (CLSM) images were obtained after incubating MCF-7 cells with the metallo-nanodrugs (equivalent concentration of Ce6: 10.0 μg mL-1). The images show that the Ce6 fluorescence is mainly located in the cytoplasm and that the fluorescence intensity gradually increased with the incubation time (Figure 4a). These results can be ascribed to gradual internalization of the metallo-nanodrugs by endocytosis and the subsequent release of Ce6 from the metallo-nanodrugs, probably inside endocytic compartments, such as the endosomes or lysosomes.27,50 The location of the released Ce6 in the cytoplasm was further confirmed by staining the cells pre-incubated with Fmoc-H/Zn2+/Ce6 for 24 h with Hoechst 33342 and Alexa 488 dyes for the cell nucleus and membrane, respectively (Figure 4b). Flow cytometry analysis revealed that the cells incubated with the metallonanodrugs (equivalent concentration of Ce6: 2.5 μg mL-1) showed higher fluorescence intensity than those incubated with unencapsulated Ce6 (Figure 4c), suggesting that the

metallo-nanodrugs can efficiently enhance the Ce6 internalization. In vitro ROS tests were carried out on MCF-7 cells pre-incubated with the metallo-nanodrugs or unencapsulated Ce6 (equivalent concentration of Ce6: 2.5 μg mL-1) for 24 h. 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was used as the fluorescent ROS probe. The results show that the ROS yield induced by the metallonanodrugs is higher than that induced by unencapsulated Ce6 (Figure 4d and S3). To demonstrate that the ROS induced by the metallo-nanodrugs are toxic to cells, the cells preincubated with Fmoc-H/Zn2+/Ce6 for 24 h were irradiated by a 635 nm laser (3 mW) for 10 min. Staining the cells with Hoechst 33342 and propidium iodide (PI), a dead-cell indicator, showed that the cells were stained only by Hoechst 33342 (blue ﬂuorescence) before irradiation, while the cells incorporated PI (green fluorescence) after irradiation (Figure 4e), which suggested cell apoptosis/necrosis was induced by phototoxicity. In contrast, the control cells not incubated with Fmoc-H/Zn2+/Ce6 were not stained by PI after the same irradiation, suggesting that the irradiation itself has negligible impact on the cells. To quantify the PDT efficacy of the


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Figure 5. In vivo blood circulation and biodistribution of the metallo-nanodrugs. (a) Concentration of Ce6 in the blood at different time points after intravenous injections of Fmoc-H/Zn2+/Ce6, Z-HF/Zn2+/Ce6, or unencapsulated Ce6. (b) Fluorescence images showing that Fmoc-H/Zn2+/Ce6 and Z-HF/Zn2+/Ce6 allow better accumulation of Ce6 in tumor sites than unencapsulated Ce6. (c) Fluorescence intensity obtained from the tumor sites in (b). (d) Fluorescence images of ex vivo organs harvested at 24 h post-injection. (e) Fluorescence intensity obtained from the ex vivo organs in (d). Error bars denote the standard deviation (n = 3). *P < 0.05 (one-way ANOVA).

metallo-nanodrugs, the cells were incubated with FmocH/Zn2+/Ce6 or Z-HF/Zn2+/Ce6 at various concentrations for 24 h and then irradiated by a 635 nm laser (100 mW cm-2) for 1 min. The cell viability was investigated after incubation for another 24 h using a standard methylthiazolyldiphenyltetrazolium bromide (MTT) assay (Figure 4f). The results revealed that the cell viability was inhibited by the combination of irradiation and the metallo-nanodrugs, especially at high concentrations of Fmoc-H/Zn2+/Ce6 or ZHF/Zn2+/Ce6, which confirmed that the cytotoxicity results from the PDT effect. Significantly, the IC50 values of FmocH/Zn2+/Ce6 (1.15 μg mL-1) and Z-HF/Zn2+/Ce6 (1.02 μg mL1 ) are much lower than that of unencapsulated Ce6 (3.83 μg mL-1), and this result suggests much better in vitro PDT efficiencies are realized for Ce6 in metallo-nanodrugs. In addition, the cells incubated with Fmoc-H/Zn2+/Ce6 or ZHF/Zn2+/Ce6 in the dark showed no significant viability changes, indicating that both Fmoc-H/Zn2+/Ce6 and ZHF/Zn2+/Ce6 are biocompatible with no inherent toxicity. The blood circulation profiles obtained by monitoring the Ce6 concentration in plasma after an intravenous injection of the metallo-nanodrugs into mice show that both FmocH/Zn2+/Ce6 and Z-HF/Zn2+/Ce6 can enhance the blood circulation time of Ce6 (Figure 5a). Fitting the profiles with a pharmacokinetic model showed that the half-lives of Ce6 in Fmoc-H/Zn2+/Ce6 (8.71 h) and Z-HF/Zn2+/Ce6 (6.33 h) are much longer than the half-life of unencapsulated Ce6 (3.69 h). The longer blood circulation times of the assembled metallonanodrugs are favorable for their accumulation at tumor sites. The in vivo distribution of the metallo-nanodrugs was

investigated in tumor-bearing mice using a fluorescence imaging system (Figure 5b). The fluorescence images demonstrate that the mice injected with the metallonanodrugs or unencapsulated Ce6 have a strong fluorescence signal throughout the whole body at 2 h after the injection, indicating a high concentration of Ce6 in the blood. Then, the fluorescence intensity of all groups gradually decreased. At 24 h post-injection, the mice injected with Fmoc-H/Zn2+/Ce6 or Z-HF/Zn2+/Ce6 showed a strong fluorescence only at the tumor sites. In contrast, no significant fluorescence was observed in the mice injected with unencapsulated Ce6 beyond 12 h post-injection. The mean fluorescence intensities of the tumor sites illustrate that Fmoc-H/Zn2+/Ce6 and ZHF/Zn2+/Ce6 are better retained in the tumors than the unencapsulated Ce6 (Figure 5c), which is presumably due to their prolonged blood circulation time and enhanced EPR effect. Ex vivo fluorescence analysis of the organs and tumors harvested at 24 h post-injection further confirmed that FmocH/Zn2+/Ce6 and Z-HF/Zn2+/Ce6 have better accumulation and retention properties than unencapsulated Ce6 (Figure 5d, 5e, and S4). In addition, strong fluorescence was observed only in tumors and livers and not other organs, indicating the selective distribution of the metallo-nanodrugs. The in vivo PDT efficacy of the metallo-nanodrugs was evaluated in tumor-bearing mice. Twenty mice were divided into four groups, and each group was injected with FmocH/Zn2+/Ce6, Z-HF/Zn2+/Ce6, Ce6, or a 5% glucose aqueous solution (control group). At 4 h post-injection, all tumor sites were irradiated by a 635 nm laser for 10 min (200 mW cm-2). After irradiation, scars were observed on the tumors in the



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Figure 6. In vivo PDT using the metallo-nanodrugs. (a) Representative photos of tumor-bearing mice at various time points after PDT. The mice were injected with Fmoc-H/Zn2+/Ce6, Z-HF/Zn2+/Ce6, unencapsulated Ce6, or a 5% glucose aqueous solution (control group). At 4 h post-injection, all tumor sites were irradiated by a 635 nm laser for 10 min (200 mW cm-2). (b) Photos of all the mice or tumors at the end of the observation showing that the skin of the mice treated with Fmoc-H/Zn2+/Ce6 and Z-HF/Zn2+/Ce6 almost recovered. (c) Tumor growth profiles during the observation. (d) Body weights of the mice during the observation. Error bars represent the standard deviation (n = 5). *P < 0.05 (one-way ANOVA).

mice treated with Fmoc-H/Zn2+/Ce6, Z-HF/Zn2+/Ce6, or Ce6 (Figure 6a), implying PDT-induced phototoxicity, while the tumors in the control group mice did not show any change, suggesting the safety of the irradiation. The scars in the groups treated with the metallo-nanodrugs gradually disappeared, and no tumor regrowth was found during the observation period (Figure 6b). In contrast, the tumors treated with Ce6 regrew several days after irradiation. The tumor growth profiles also show that the metallo-nanodrugs induced effective photodynamic tumor ablation, while unencapsulated Ce6 only partially inhibited the tumor growth (Figure 6c). The body weights of the mice in all groups were not significantly different (Figure 6d), which indicated that the metallonanodrugs are biocompatible. In addition, the internal organs, including the heart, liver, spleen, lung, and kidneys, harvested at the end of the observation period did not show cell death or inflammation signals (Figure S5). We further investigated the therapeutic effect of the individual irradiation, the metallonanodrugs without the irradiation, and the peptide-Zn2+ assemblies without Ce6. The tumor volumes and body weights suggest that these treatments exhibit no therapeutic effect, confirming that the therapeutic effect can only be induced by PDT through the combination of the irradiation and the metallo-nanodrugs (Figure S6). These results demonstrate that Fmoc-H/Zn2+/Ce6 and Z-HF/Zn2+/Ce6 are efficient metallo-nanodrugs with good biocompatibility.

Conclusion In summary, we developed a multicomponent self-assembly strategy based on cooperative coordination to rationally engineer metallo-nanodrugs for efficient antitumor photodynamic therapy. We have demonstrated that metallonanodrugs can be readily formed by cooperative coordination of small peptides and photosensitizers in the presence of metal ions. The resulting metallo-nanodrugs have well-defined nanostructures, uniform size distributions, and impressive photosensitizer loading capacities and encapsulation efficiencies. Importantly, the metallo-nanodrugs feature both robust stabilities under normal physiological conditions and burst responsiveness to pH and glutathione (GSH) level variations. Due to the integration of robust blood circulation and targeted burst release, metallo-nanodrugs have a prolonged blood circulation lifetime, enhanced accumulation in tumors, and improved antitumor efficacy compared to those of unencapsulated photosensitizers. These properties guarantee efficient photodynamic ablation of tumors without off-target side effects. Compared to existing nanodrugs, metallo-nanodrugs have unique advantages: First and foremost, metallo-nanodrugs are formed by the combination and cooperation of coordination and multiple non-covalent interactions, leading to the facile integration of robust blood circulation and targeted burst


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release; Second, the preparation approach is very simple and readily manipulated; Third, metallo-nanodrugs provide extremely high loading capacities and encapsulation efficiencies; Finally, metallo-nanodrugs are highly biocompatible because all the building blocks are biologically benign materials. These advantages reveal that multicomponent coordination self-assembly is a promising strategy to fabricate metallo-nanodrugs for improved antitumor therapy through spatiotemporal, controlled delivery of photosensitizers. Because many antitumor drugs contain metal-binding motifs, this multicomponent coordination self-assembly strategy can be explored for other drugs to improve their antitumor efficacy. The morphology, architecture, and functionality of the metallo-nanodrugs can be further tailored by changing the type and ratio of the peptides and metal ions to facilitate on-demand manipulation and functionalization of metallo-nanodrugs. This suggests that a supramolecular strategy based on multicomponent cooperative coordination could be further designed and optimized to advance biomedical nanotechnology and clinical translation of metallonanodrugs for diagnosis and treatment of various diseases.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: x. Materials and instruments, methods, supporting Figures S1-S6, and supporting Tables S1-S3.

AUTHOR INFORMATION Corresponding Author *[email protected]

ORCID Qianli Zou: 0000-0003-0464-4156 Xuehai Yan: 0000-0002-0890-0340

Author Contributions #

S.L. and Q.Z. contributed equally.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work is dedicated to the memory of Prof. Helmuth Möhwald. We acknowledge financial support from the National Natural Science Foundation of China (Project Nos. 21522307, 21473208, 21773248, and 21603233), the Talent Fund of the Recruitment Program of Global Youth Experts, the Key Research Program of Frontier Sciences of the Chinese Academy of Sciences (CAS, Grant No. QYZDB-SSW-JSC034), and the CAS President’s International Fellowship Initiative (2017DE0004 and 2017VEA0023).

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