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Biological and Medical Applications of Materials and Interfaces

A Smart Photosensitizer: Tumor-triggered Oncotherapy by Self-Assembly Photodynamic Nanodots Yuhua Jia, Jinyu Li, Jincan Chen, Ping Hu, Longguang Jiang, Xueyuan Chen, Mingdong Huang, Zhuo Chen, and Peng Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 13 Apr 2018 Downloaded from http://pubs.acs.org on April 13, 2018

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ACS Applied Materials & Interfaces

A Smart Photosensitizer: Tumor-triggered Oncotherapy by Self-Assembly Photodynamic Nanodots Yuhua Jia 1,2, Jinyu Li *3, Jincan Chen1, Ping Hu1, Longguang Jiang3, Xueyuan Chen1, Mingdong Huang1,3, Zhuo Chen*1, Peng Xu*1

1

State Key Laboratory of Structural Chemistry and CAS Key Laboratory of Design and Assembly

of Functional Nanostructures, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, Fujian 350002, P.R. China 2

College of Life Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002,

P.R. China 3

College of Chemistry, Fuzhou University, Fuzhou, Fujian, 350116, P.R. China

KEYWORDS: self-assembly nanodots, photosensitizers, photodynamic therapy, photobleaching, breast cancer models, molecular dynamics.

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ABSTRACT: Clinical photosensitizers suffer from the disadvantages of fast photobleaching and high systemic toxicities due to the off-target photodynamic effects. To address these problems, we report a self-assembled pentalysine-phthalocyanine assembly nanodots (PPAN) fabricated by an amphipathic photosensitizer-peptide conjugate. We triggered the PDT effects of photosensitizers by precisely controlling the assembly and disintegration of the nanodots. In physiological aqueous conditions, PPAN exhibited a size-tunable spherical conformation with a highly

positive shell of the polypeptides and a hydrophobic core of the π-stacking Pc moieties. The assembly conformation suppressed the fluorescence and the reactive oxygen species (ROS) generation of the monomeric photosensitizer molecules (mono-Pc), and thus declined the photobleaching and off-target photodynamic effects. However, tumor cells disintegrated PPAN and released mono-Pc molecules, which performed fluorescence and the photodynamic effects for the detection and elimination of tumor tissues. The molecular dynamics (MD) simulation revealed the various assembly configurations of PPAN and illustrated the assembly mechanism. In cellular level, PPAN exhibited remarkable phototoxicity to breast cancer cells with the IC50 values in low nanomolar range. By using a subcutaneous and an orthotopic breast cancer animal models, we also demonstrated the excellent antitumor efficacies of PPAN in vivo.


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INTRODUCTION Photodynamic therapy (PDT) is a promising therapeutic modality for various malignant cancers.1-3 Compared to surgery, chemo- and radio-therapies, which bring severe suffering to patients, PDT causes much milder systemic damage for the non-invasive nature.4 PDT eliminates tumors through the generation of the highly cytotoxic reactive oxygen species (ROS) and the shutdown of angiogenesis.5 In addition, in contrast to chemotherapies, which suppress the natural immune systems, PDT stimulates immune responses to facilitate the clearance of cancers.6-7 Currently, clinically used photosensitizers are all porphrin-, chlorin-, and phthalocyaninederivatives with marginal or axial modifications.8-10 The solubility and biocompatibility problems of these aromatic macrocyclic compounds are generally compromised by the addition of solubilizers or chemically modified with hydrophilic groups. A paradox for the design of medicinal photosensitizers is to develop aggregated or depolymerized molecules to fit the physiological and pathological conditions. The aggregation inevitably declines the photodynamic effects of generating ROSs, and thus impairs the antitumor efficacies of the photosensitizers.11-12 In contrast, the depolymerized photosensitizers always suffer from the insufficient stability because of the photo bleaching.12 More importantly, the bioavailability of photosensitizers in the monomeric form is normally low due to the short circulating time and the fast renal clearance in vivo. In addition, the off-target accumulations of the photosensitizer monomers is risky to cause unnecessary systemic damages, including skin burns.8 An ideal mode for medicinal photosensitizers is in the aggregated configuration for storage avoiding photo bleaching, and circulate in the aggregation form to reduce the renal clearance and systemic toxicities in vivo, while depolymerize and perform photodynamic effects when approaching tumor sites. For this purpose, in this study, we proposed a unique strategy to specifically trigger the photodynamic effects in tumor sites by precisely controlling the aggregation and depolymerization of photosensitizers in physiological and pathological conditions. We

hereby

report

a

tumor-triggered

photodynamic

therapy

by

self-assembly

pentalysine-phthalocyanine assembly nanodots (PPAN), which are fabricated by an amphipathic conjugate of a pentalysine peptide and a phthalocyanine photosensitizer (Figure 1A). PPAN were designed to trigger the photodynamic effects specifically at tumor sites by precisely controlling the release of monomeric pentalysine phthalocyanine (mono-Pc) molecules. In



physiological aqueous conditions, mono-Pc molecules assemble into PPAN by embedding the hydrophobic phthalocyanine (Pc) moieties interiorly while exposing the positively charged pentalysine moieties on surface. This spherical assembly configuration protects the Pc rings from photobleaching. Besides, in circulating systems, the assembly configuration of PPAN significantly increases the molecular sizes and prolongs the circulating period of the photosensitizer in vivo. In addition, assembly passivates the fluorescence and photodynamic effects of mono-Pc molecules, reducing the consumptions and the systemic toxicity in circulating systems. The solvent-exposed pentalysine moieties facilitate PPAN targeting the negatively charged surfaces of tumor cells through electrostatic attractions.13 Once contacting with tumor cells, the amphipathic lipid bilayers of biological membranes depolymerize PPAN and release the mono-Pc molecules, which perform the photodynamic effects and fluorescence for the elimination and detection of cancers (Figure 1A).

Figure 1: A. Tumor-disintegrable self-assembled PPAN: In physiological aqueous conditions, mono-Pc molecules assemble into a spherical conformation with a positive shell and a π-π stacking Pc core. The assembled conformation inactivated the photodynamic effects of mono-Pc. Tumor cells disintegrate PPAN to trigger the release of mono-Pc molecules, which perform photodynamic effects to eliminate the cancer cells; B: The SEM topographical images of PPAN; C: The AFM images of PPAN; D: The height profile of a single dot along the white line in the


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AFM image. E-F: the uv-vis absorption spectra (E) and fluorescence spectra (F) of PPAN in DMSO, PBS and PBS with 1% SDS respectively; G. the hydrodynamic diameters determined by DLS in buffers at varied pH values (2.2 - 8.0).

RESULTS Design and preparation of PPAN In aqueous conditions, the amphipathic molecules have the tendency of self-assembly, normally in the configuration of encapsulating the hydrophobic parts and exposing the hydrophilic parts. By unsymmetrically modified with polypeptides, the aromatic macrocyclic photosensitizers will be amphipathic with a hydrophobic photosensitizer terminus and a hydrophilic polypeptide terminus. Thus, we designed a self-assembly photosensitizer-peptide molecule by conjugating phthalocyanine (Pc) and pentalysine. Phthalocyanine zinc was selected because of the deeper therapeutic depth and the higher ROS yields compared to porphrin- and chlorin- photosensitizers.14-15 The highly positive pentalysine was selected as the hydrophilic moiety in order to target the negatively charged surface of tumor cells due to the overexpression of sialic acid.13 With this purpose, we synthesized a pentalysine-phthalocyanine conjugate with high purity through the routine solid-phase peptide synthesis as reported previously (Figure S1).16 The peptide-phthalocyanine assembly nanodots (PPAN) were prepared by firstly dispersing the pentalysine-phthalocyanine conjugate in DMSO followed by the dilution with 100-fold volume of distilled water (ds-water). The DMSO was next discarded via the dialysis against proper solvents (PBS or ds-water). The monomeric pentalysine-phthalocyanine conjugate (mono-Pc) then self-assembled to the spherical conformation of PPAN with a core of the

π-stacking Pc rings and a highly positive surface of pentalysines (Figure 1A). Scanning electron microscopy (SEM) and atom force microscopy (AFM) data demonstrated that PPAN were in a spherical conformation with the average sizes of 27.4 and 35.2 nm, respectively (Figures 1B and 1C), mainly consistent with the hydrodynamic diameters of 37.8 nm determined by dynamic light scatter (DLS) at pH 7.4 in PBS (Figure 1G). The average zeta potential of PPAN was 18.7 mV confirming the positive surface (Figure S4).



The mechanism of self-assembly of PPAN

Figure 2: The self-assembly mechanism of PPAN revealed by MD simulation. A: The schematic diagram of the assembly process of PPAN. The Pc moiety and the pentalysine moiety are represented as yellow and blue ellipse, respectively. B: The 3-D structures of the oligomers demonstrated in panel A. The mono-Pc molecules forming Dimer 1 and Trimer are showed in cyan and yellow sticks, respectively. The coordinated Zn was shown in magenta sphere. Other atoms are colored as cyan/yellow (C), red (O), blue (N), and white (H). Polar interactions intraand inter- mono-Pc molecules are shown in black dash lines.

Self-assembly process of PPAN was revealed by molecular dynamics (MD) simulations. For the initial structures of the simulation, five mono-Pc molecules were randomly inserted into a water box with edge lengths of 10 × 10 × 10 nm3. After energy minimization, heating and equilibration, 500 ns-long all-atom MD simulations were performed on the isothermal–isobaric (NPT) ensemble at a constant temperature of 300 K (see more details in the Experimental Section). The representative snapshots of the system obtained from MD simulations were shown in Figures 2 and S3. In the beginning, all the five mono-Pc molecules were monomeric. At 4 ns, two mono-Pc molecules quickly aggregated to Dimer 1 through the π-aggregates, while the other 3 molecules were still monomeric. At 209 ns, another dimer (Dimer 2) formed and stabilized through intermolecular polar interactions. At 276 ns, Dimer 2 was converted to a more stable π-aggregates aggregation conformation. At 384 ns, a trimer formed based on the π-π stacking of


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the Pc rings of Dimer 2 and the residual monomer. We then observed the formation of pentamers after 400 ns. Specifically, the MD simulations revealed that the pentamers can be organized via two distinct and progressive assembly patterns (Pentamer 1 and Pentamer 2 in Figure 2) during the last 100 ns (400-500 ns). From 400-435 ns, Pentamer 1 firstly appeared and was stabilized by the inter-oligomer salt bridges between the pentalysines moieties of Dimer 1 and Trimer. In contrast to the polar driving force of Pentamer 1, the Pc rings from the trimer and dimer in Pentamer 2 formed non-polar parallel staggered stacking interactions with a dihedral angle between stacking planar Pc groups varying from 44.2° to 63.8° (from 435 to 500 ns), a conformation similar to that of 12 planar 5,6-indolequinone tetramers observed before.17 The larger occupancy of Pentamer 2 with respect to Pentamer 1 during the MD simulations indicates that Pentamer 2 may be the more favorable assembly pattern in aqueous solution. This is probably due to the need to 1) minimize the electrostatic repulsions between lysine amino acids from different oligomers and, 2) maximize the stacking interactions between the hydrophobic Pc groups. Therefore, the stacking interactions may be the major driving force for the self-assembly of mono-Pc molecules in aqueous solution. To summarize, mono-Pc molecules can aggregate through either the polar interactions of the pentalysines chains or the π-stacking of the Pc rings. However, the stable oligomers demonstrate a basic conformation with a core of the stacking Pc rings and a shell of solvent exposing pentalysines, which is in agreement with our expectation (Figure 1A).

Self-assembly changed the photophysical properties of photosensitizer As reported, in uv-vis absorption spectra, Pc molecules in the monomer configurations show sharp and strong Q-band absorbance around 680 nm. In contrast, aggregated Pc molecules show board but much weaker Q-band absorbance with the maximum absorbance around 630 nm.18 In DMSO, PPAN demonstrated a strong absorbance peak at 678 nm (Figure 1E), showing that most Pc molecules were in the monomer configuration. In contrast, in PBS, Pc molecules were mostly in the assembly configuration because of the board Q-band absorbance around 630 nm. When PBS was supplemented with 1% Sodium dodecylbenzene sulfonate (SDS), the assembly absorbance (630 nm) decreased while the monomeric absorbance (670 nm) increased, indicating the depolymerization of PPAN in the presence of the detergent. We next determined the



fluorescence spectra of PPAN in DMSO, PBS, and PBS with 1% SDS at λex = 610 nm (Figure 1F). In DMSO, mono-Pc demonstrated strong fluorescence emission ranging from 650 to 800 nm with the maximum emission at 690 nm. However, this fluorescence was mostly suppressed when PPAN were dispersed in PBS, which is likely due to the self-quenching by the π-stacking Pc rings. In PBS with 1% SDS, the fluorescence emission partially restored due to the depolymerization. Thus, the self-assembly configuration of PPAN in aqueous conditions significantly changes the uv-vis absorption and quenches the fluorescence of the mono-Pc molecules.

The pH-mediated assembly configurations The hydrodynamic diameters (HDs) of PPAN are sensitive to the environmental pH values. DLS analysis showed that the HDs of PPAN (10.6-67.8 nm) increased corresponding to the increase of pH values (2.2-8.0) (Figure 1G). The sensitivity of HDs to the pH values is likely because of the different protonation levels of the pentalysines moiety at different pH values. In acidic conditions, fewer mono-Pc molecules aggregated in one single nanodot due to the higher electrostatic repulsion among the highly protonated pentalysine moieties. In contrast, in alkaline conditions, lower electrostatic repulsion allows more mono-Pc molecules assembled in one spherical nanodot. We also found that PPAN showed a pH-dependent Q-band absorbance (550-750 nm): the higher pH values corresponded to the higher absorbance (Figure S5), which is in good agreement with the fact that higher pH values led to larger sizes. We next evaluated the influence of the ionic strength on the assembly configuration of PPAN (Figure S6). The sizes of PPAN showed very minor changes in phosphate buffer pH 7.4 with NaCl concentrations ranged 30-500 mM. However, the sizes declined when the NaCl concentrations increased to the range of 1000-4000 mM. This result indicates that ionic strength can only slightly influence the assembly configuration of PPAN.

The disintegration of PPAN We have demonstrated above that the assembly configuration of PPAN was disintegrated in organic solvents or in aqueous solutions containing detergents. In the solvent of DMSO-PBS mixture, PPAN showed increased monomeric absorbance at 680 nm with the increase of DMSO


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concentration (Figure 3A), indicating the higher depolymerization in solvent with higher ratio of organic solvents. Similarly, PPAN demonstrated a DMSO-dependent increase in fluorescence emission at λex = 610 nm because of the disintegration (Figure 3B). Next, we evaluated the disintegration of PPAN in the presence of tumor cells. We measured the uv-vis absorption spectra of PPAN with and without incubation with tumor cells (Figure 3C). In PBS without cells, PPAN mainly showed the specific absorbance of the assembly conformation (630 nm). In contrast, at the same concentration, PPAN demonstrated remarkably enhanced monomeric absorbance (680 nm) and declined assembly absorbance (630 nm) in the presence of tumor cells, suggesting that contacting with tumor cells disintegrated PPAN and released the mono-Pc molecules.

Figure 3: Disintegration of PPAN nano dots. A-B: the uv-vis absorption spectra (A) and fluorescence spectra (B) of PPAN in solvents containing different concentrations of DMSO. C. the Q-band absorbance of PPAN in the presence and absence of 4T1 cells; D. the ROS generations of PPAN and control Pc in PBS, PBS containing 75% FBS, and 4T1 cells, the ROS generation was presented as the fluorescence of the ROS probe per pmol Pc. E. the confocal fluorescent imaging of PPAN incubated with in 4T1 cells in the absence and presence of 25% and 50% serum. The PPAN fluorescence intra and extra cells was marked with yellow and green arrows, respectively.



The tumor-triggered photodynamic effects and fluorescent emission of PPAN The antitumor efficacies of PDT agents are largely dependent on the yields of ROS generation. We next compared the ROS generation of PPAN in PBS and PPAN internalized in tumor cells by using 2’,7’-dichlorofluorescin diacetate (DCFH-DA) as a fluorescent ROS sensitizer (Figure 3D). In PBS, the low ROS generation consisted with the fact that the assembly conformation of PPAN suppressed the photodynamic effects. In contrast, in tumor cells, the released mono-Pc demonstrated significantly enhanced ROS generation, which was also in agreement with the disintegration of PPAN in tumor cells. This result indicates that the photodynamic effects of PPAN can be triggered by contacting with tumor cells. For comparison, we also determined the ROS generation of PPAN in the presence of 75% fetal bovine serum (FBS) to mimic the circumstance in vessels. The ROS generation in the presence of serum is slightly enhanced compared to that in PBS, while much lower than that of the photosensitizers internalized in tumor cells. The fluorescence of PPAN in serum and in tumor cells were compared through the fluorescent confocal microscopy with the same excitation voltage and offset value (Figure 3E). Breast cancer cells, 4T1, were incubated with 1 μM PPAN in PBS containing 0, 25%, and 50% FBS. The and extracellular (green arrows in Figure 3E) fluorescent signals simulated the PPAN fluorescence in blood, respectively. No extracellular PPAN fluorescence was observed in PBS without serum, consistent with the above result that the assembly configuration completely suppressed the fluorescence of Pc. In contrast, PPAN internalized in 4T1 cells exhibited strong fluorescence indicating the high-degree disintegration of PPAN in tumor cells. The extracellular fluorescence slightly enhanced with the increase of serum, suggesting that serum can slightly disintegrate the assembly configuration. However, the extracellular fluorescence was still much weaker than the intracellular fluorescence, which, consistent with the ROS generation determination, indicates that the macrobiomolecules (proteins, lipids, nucleic acids) in blood can also partially disintegrate the self-assembly configuration of PPAN. However, the disintegration in blood is much milder than that in tumor cells.


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Photodynamic antitumor effects and bio-safety in vitro

Figure 4: Antitumor efficacies in vitro. A. the phototoxicities of PPAN and control Pc to breast cancer cell line, 4T1, and healthy cell line, L-02; B. the percentages of the apoptotic (red) and necrotic (blue) cells after PDT with 0, 10, 33, and 10 nm PPAN.

The antitumor efficacy and the safety of PPAN were evaluated by determining the phototoxicities to the murine breast cancer cell line, 4T1, and a healthy hepatic cell line, L-02 (Figure 4A). β-carboxyphthalocyanine zinc, which lacks of the pentalysine moiety and thus has no tendency of assembly, was set as the control photosensitizer. Neither PPAN nor control Pc showed measurable cellular toxicity to 4T1 or L-02 cells without illumination (Figure S8). With the light dosage of 1.5 J/cm2, both PPAN and control Pc demonstrated remarkable antitumor efficacies (Figure 4A). PPAN (IC50 = 15 ± 2.3 nM) demonstrated 3-fold higher phototoxicity to 4T1 cells than the control Pc did (IC50 = 47 ± 1.8 nM), which was consistent with the 3-fold higher cellular uptake of PPAN in 4T1 cells (Figure S7). Control Pc showed comparable phototoxicity in both 4T1 cells and the healthy cell line, L-02, indicating that normal Pcs lack the selectivity between tumor cells and healthy cells. In contrast, PPAN was much more specific to tumor cells because PPAN showed much lower phototoxicity to healthy cells than that to tumor cells. The selectivity of PPAN is likely due to the electrostatic attractions with the negative sialic acid overexpressed on tumor surfaces.

The apoptosis caused by PPAN was detected through the annexin V-FITC / propidine iodide (PI) double stainings using flow cytometer (Figure 4B and S9). As indicated, the antitumor effects of PPAN were through both the apoptosis and necrosis pathways. The control group without PPAN treatment showed a 97.4% viability, indicating that the cells were in good conditions. When treated with 10, 33, and 100 nM of PPAN, the ratios of the



apoptotic and necrotic cells showed the dose-dependent enhancement. Notably, treatment with PPAN caused higher amount of necrotic cells than that of apoptotic cells. In addition, we evaluated the influence of PDT with PPAN on cell cycles and found that the amounts of the cells in G1, S and G2 stages were mostly identical with and without the treatment of PPAN (Figure S10), demonstrating the genetic safety.

Subcellular Localization of PPAN

Figure 5: Confocal fluorescence microscopy of PPAN co-stained with organelle probes in MDA-MB-231. PPAN were co-stained with the nuclei probe, DAPI (A), the lysosomes probe, LysoTracker® Red (B), and the mitochondria probe, MitoTracker® Green (C), respectively. The first lane (red) shows the fluorescent images of PPAN, the second lane (blue and green) shows the fluorescent images of the organelle probes, the third lane shows the overlaid images. The overlaps of PPAN and lysosomes / mitochondria were quantified in Figure S11.

To further investigate the mechanism of the antitumor effects of PPAN, the sub-cellular localization was determined via a laser scanning confocal microscope (Figure 5). Breast cancer cells, MDA-MB-231, were co-stained with PPAN and fluorescent organelle markers before examination. The optical images showed that PPAN did not penetrate the nuclei membrane, avoiding the risk of genetic damages. PPAN was found to be localized in both mitochondria and lysosomes, with higher accumulation in lysosomes (Figure S11).


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Photosensitizers accumulating in mitochondria are believed to perform much higher photodynamic effects than those accumulating in other organelles.19-20 Besides, the damage of mitochondria or releasing ROS in mitochondria can trigger the apoptotic cascades of tumor cells.21-22 Thus, the higher accumulation in lysosomes than in mitochondria was in good agreement with the fact that PPAN caused higher ratio of necrosis than that of apoptosis.

Anti-tumor effects and biodistribution in a subcutaneous breast cancer animal model

Figure 6: Anti-tumor effects and biodistribution in a subcutaneous breast cancer animal model. A-B. Tumor volumes (A) and body weights (B) were measured daily after the PDT treatment with saline, 0.019 mg/kg control Pc, 0.042 mg/kg and 0.126 mg/kg PPAN. Mice were illuminated on Day 1 24 hr after intravenous dosing; C-D. Tumor weights (C) and photographs (D) of the tumors resected on Day 8. E. Imaging graphics of PPAN in 4T-1 bearing mice at 12 h, 24 h, 48 h, and 72 h after intravenous injections. The scale ruler indicates the intensity of the fluorescent energy. F. Concentrations of PPAN in the organs of 4T1-bearing mice. The values were represented as Mean ± SEM, *P < 0.05, **P < 0.01, ***P300-fold longer blood half-life and >700-fold longer body half-life than QDs with HDs < 5.5 nm did.34 In our study, in physiological condition (pH 7.4), PPAN in the assembly configuration showed HD of 37.8 nm, which was


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significantly larger than that of the mono-Pc molecules (HD = 2-3 nm based on the MD simulation), and thus remarkably prolonged the circulating period in vivo (Figure 6E and 6F). In addition, the pH-tunable sizes of PPAN led to the much faster diffusion in tumor tissues than that in healthy tissues. Compounds or materials with amine groups are majorly sensitive to the environmental pH values because of the protonation. In this study, the HDs of PPAN demonstrated lower HDs in acidic conditions (Figure 1G). Tumor tissues have been confirmed to be in a relatively acidic circumstance due to the overexpression of sialic acid. At acidic conditions, PPAN showed a diameter of 11.7 nm, which facilitated the diffusion in tumor tissue. At healthy tissues (pH = 7.4), PPAN showed a diameter of 37.8 nm, which were much less diffusive. Thus, PPAN can infiltrate and accumulate in tumor tissues much faster than in healthy tissues, indicating the specificity to tumor tissues over healthy tissues.

Furthermore, the photodynamic effect of PPAN was triggered by the therapeutic target, the tumor cells. Therefore, the occurrence of photodynamic effects was precisely controlled in tumor sites. The assembly conformation of PPAN inactivated the fluorescence and photodynamic effects of mono-Pc, which prevented the photobleaching and the off-target consumption. One important reason of the consumption of photosensitizers is the large amount of widely distributed antioxidants in vivo, which neutralize the ROSs generated by the off-target photodynamic effects. Fan et al loaded photosensitizers with MnO2-nanosheets to reduce the physiological glutathione level in vivo.35 Our strategy is to embed the photosensitive moiety in the sphere center of PPAN nanodots in order to prevent contacting with antioxidants in circulating system. When accumulating in tumor tissues and contacting tumor cells, self-assembled conformation of PPAN was disintegrated. The released mono-Pc then performed the fluorescence for detection and photodynamic effects to eliminate cancer cells. Thus, the tumor-triggered PDT is able to efficiently reduce the photobleaching and systemic damage.

CONCLUSIONS In brief, we developed a tumor-triggered photodynamic therapy by employing the peptide-phthalocyanine assembly nanodots (PPAN), which was fabricated by the monomers of



an amphipathic pentalysine-phthalocyanine zinc conjugate (mono-Pc). In physiological conditions, PPAN demonstrated a spherical conformation with a highly positive shell and a hydrophobic core of the π-π stacking Pc moieties. The sizes of PPAN can be mediated by the environmental pH values. At acidic conditions in tumor tissues, PPAN showed a diameter of approximate 10 nm which facilitated the diffusion in tumor cells. Because of the self-assembly conformation, the fluorescence and photodynamic effects of mono-Pc molecules were mostly inactivated avoiding photobleaching and systemic damages. However, once internalized in tumor cells, PPAN disintegrated, and thus mono-Pc molecules were released. The restored fluorescence and photodynamic effects were used for the detection and elimination of cancer cells. In cellular level, PPAN exhibited remarkable phototoxicity to breast cancer cells and much lower phototoxicity to healthy cells. We further demonstrated that the released mono-Pc molecules were localized in mitochondria and lysosomes and caused the cell death through the combination of apoptotic and necrotic pathways with no obvious influence on the cell cycles. Furthermore, we established a subcutaneously grafted and an orthotopic animal models of breast cancer to evaluate the anti-tumor efficacy of PPAN in vivo. Treating with PPAN efficiently suppressed the tumor growth in both animal models at the low dosages of illumination and the photosensitizer. Thus, tumor-disintegrable PPAN we developed might provide a new approach to improve the biocompatibility of aromatic photosensitizers and a novel strategy to precisely control photodynamic effects in tumor sites.

EXPERIMENTAL SECTION Synthesis, purification, and characterization of mono-Pc and PPAN. Pentalysine-phthalocyanine conjugate (7 in Figure S1, mono-Pc) was synthesized as described in our previous study (Figure S1).16 Briefly, β-Carbonylphthalocyanine (3, Control Pc) was synthesized by the statistical condensation of trimellitic anhydride 1 with phthalic anhydride 2 as described in our previous work.36 The pentalysine with the exposed N-terminus and the t-butyloxy carbonyl (Boc) side chains were coupled to Wang resin through the C-terminus carbonyl group (5). 3 was then coupled with 5 through the amidation between the carboxyl group of 3 and the amine of 5 in the presence of hexafluorophosphate tetramethylurea (HBTU) and N’N-Diisopropylethylamine (DIEA). The resin coupled mono-Pc (6) was cleaved from the resin


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by incubation with 95% trifluoroacetic acid for 3 h and purified through the synthetic HPLC chromatography, obtaining mono-Pc (7) with a single component and the purity >95% (Figure S2). Analytical HPLC was performed on a C18 RP HPLC system (Dalian Elite Analytical Instruments Co. Ltd., Dalian, China; column: SinoChrom ODS-BP, 250×4.6 mm, 5 µm) using a linear gradient of 50, 100% CH3OH/CH3CN (1:1 v/v) at a flow rate of 1ml min-1 The pentalysine-phthalocyanine assembly nanodots (PPAN) were prepared by firstly dispersing the lyophilized mono-Pc in 50 µl DMSO to the concentration of 10 mM followed by the dilution with 99-fold volume of distilled water (ds-water) until the concentration of 100 µM. The DMSO was then discarded via the dialysis against proper solvents (PBS or ds-water) overnight at the room temperature. Surface topography of the self-assembly configuration of PPAN was imaged by using an atomic force microscope (AFM, Nanoscope III, Digital Instruments Co.) in contact tapping mode. Scanning electron microscope (SEM) images were obtained on a JSM-6701F field emission scanning electron microscope (FE-SEM) at 5–10 kV. The particle sizes and zeta potentials of PPAN in aqueous solvents were analyzed using dynamic light scattering (DLS) instrument (ZETASIZER 3000, Malvearn Instruments, Ltd.) at the concentration of 2 µM.

Molecular dynamics simulations. The geometry of mono-Pc was optimized by using GAMESS37 with B3LYP functional38. The basis sets of effective core potential LanL2DZ39 and 6-31G(*)40 were used on zinc and other atoms, respectively. Five optimized mono-Pc molecules were randomly inserted into a box of explicit water (100 Å × 100 Å × 100 Å) and 150 mM NaCl. Chloride ions were added to counterbalance the charge of mono-Pc molecules. The system contained 88,648 atoms in total. It was underwent MD simulations using the GROMACS 4.6.5 code

41

on GPU clusters. The

general AMBER force field (GAFF) 42, Åqvist potential 43 and TIP3P model 27 were used for the mono-Pc, ions and water molecules, respectively. All bond lengths were constrained by LINCS algorithm

44

. Periodic boundary conditions were applied. Electrostatic interactions were

calculated using the Particle Mesh-Ewald (PME) method

45

, and van der Waals and Coulomb

interactions were truncated at 10 Å. The system underwent 1,000 steps of steepest-descent energy minimization with 1,000 kJ·mol−1·Å−2 harmonic position restraints on the mono-Pc



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molecules, followed by 2,500 steps of steepest-descent and 2,500 steps of conjugate-gradient minimization without restraints. The system was then gradually heated from 0 K up to 300 K in 20 steps of 2 ns. After that, 500 ns-long productive MD simulations were carried out in the NPT ensemble. To assess the convergence of the simulated trajectory we considered the projection of the trajectory on the top essential dynamical spaces obtained from a standard covariance analysis. Following Hess’s criterion

46

, these projections were next compared with those expected for a

random reference. The observed negligible overlap (i.e. cosine content close to 0, data not shown) confirms a posteriori adequate sampling of the complex conformations around the equilibrium position. Hydrogen bond interactions were defined to be present if the atomic distance between the acceptor and donor atoms is below 3.5 Å and the angle among the hydrogen-donor-acceptor atoms is below 30 degree. Hydrophobic interactions were defined to be present if the center-of-mass distance between side chains are smaller than 4.5 Å

47

. The dihedral angles

between stacking planar Pc groups were calculated with VMD 1.9.2 and Tcl/Tk script.

Photophysical properties of PPAN. The uv-vis absorption spectra of PPAN in different solvents were recorded from 500 to 800 nm with an interval of 1 nm using a quartz cuvette with 1 cm path length on a Lambda-35 UV/Vis spectrometer (PerkinElmer, Waltham, MA). The fluorescence spectra (from 640 nm to 800 nm) of PPAN in different solvents were measured on a UV/V/NIR/MIR Fluorescence Spectrometer FSP920 (Edinburgh Instruments, Kirkton Campus, UK) with the excitation wavelength of 610 nm.

Phototoxicity and dark toxicity in vitro. The phototoxicites and dark toxicities of PPAN and control Pc were determined using the MTT Cell Viability Kit (Roche, Genermany) according to the instruction of the manual. Aliquots of exponentially growing cells (1×105 cells/ml) were placed in 96-multiwell plates at a volume of 100 µl per well and incubated for 12-16 h. The cells were incubated with complete medium with various concentration of photosensitizers (10-5.5, 10-6, 10-6.5, 10-7, 10-7.5, 10-8, 10-8.5, 10-9, 10-9.5 M) for 2 h. 1% DMSO was used to facilitate the dispersion of control Pc. One control column in the plate was filled with culture medium as a blank. The cells were washed with sterile PBS twice


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and filled with 200 µl fresh culture medium. The cells were then illuminated at a light dosage of 1.5 J /cm2 using a 680 nm LED light source for 37 s. After illumination, cells were continuatively cultivated for another 12 h. Cellular viability was then measured by adding 10 µl MTT solution (5 mg/ml in PBS) to each well followed by incubation for 4 h at 37 oC. The supernatant was subsequently removed, and 100 µl dimethyl sulfoxide (DMSO) was refilled to solubilize the formazan crystals. Then samples were measured on a microplate reader by recording the absorbance at 570 nm. The cell viability of treated samples was then obtained by comparison with the photosensitizer-free columns. Dark toxicity was measured in parallel without irradiation.

Antitumor efficacy in a subcutaneous breast cancer animal model. Approximately 107 viable cells, suspended in 200 µl sterilized saline, were inoculated into the right flank of each mouse. Tumor sizes were monitored everyday with digital caliper measurements starting on detection of palpable tumors. Tumor volumes were calculated using the modified ellipsoid formula 1/2*(length*with2). When the tumors volumes reached 80-100 mm3 in typically 3-5 days after inoculation, the tumor-bearing mice were randomly divided into four groups (8 mice per group) with the equivalent average starting tumor size (80-120 mm3) and bodyweight (20-25 g). PPAN (0.126 mg/kg, 0.042 mg/kg), control Pc (0.0187 mg/kg) in sterile saline were injected into the four groups via tail vein. One group treated with sterile saline was set as control. After 24 h post injection, mice were exposed to a 680 nm light source for 3 min to a light dosage of 30 J/cm2. Tumor volumes and body weights were recorded daily for 8 days after illumination. On day 8, the tumors were resected after the mice were sacrificed. The resected tumors were weighted and photographed.

Ex vivo imaging and biodistribution of PPAN The 4T1 tumor-bearing mice were randomly divided into 5 groups (8 mice per group) with the equivalent average starting tumor sizes of 350−400 mm3. PPAN at dosage of 10 µmol/kg in a volume of 8 ml/kg dispersed in saline were treated through tail intravenous injection at different time points (12 h, 24 h, 48 h, 72 h). A saline-treated group was set as a negative control. After mice were anesthetized and sacrificed, the organs (tumor, liver, kidney, lung, brain, and heart) were resected and imaged by using a fluorescent molecular tomography FMT 2500TM LX



instrument (PerkinElmer, Waltham, MA, U.S.) with a 680 nm laser diode for excitation. Fluorescence emission with longer wavelength (690−740 nm) was collected. The collected images were reconstructed by the software TrueQuant version 3.0 (PerkinElmer. Waltham, MA, USA). Merged graphics of transparent and fluorescent fields were showed in Figure S12. The concentrations of PPAN accumulated in different organs were quantified by determining the ZnPc fluorescence in the lysates of the organs. Detailedly, 100 mg of each resected organ was homogenized and sonicated with 1 mL of lysis buffer (0.1 M NaOH and 1% SDS) for 10 min. The homogenates were centrifuged at 4 °C, and the photosensitizer concentration in the supernatant was determined by the fluorescence of ZnPc (λex 610 nm and λem 690 nm) with the calibration of a standard curve.

Antitumor efficacy in an orthotopic breast cancer animal model. To further determine the anti-tumor efficacy of PPAN in vivo, we established an orthotopic breast cancer model by grafting 4T1 cell line containing the luciferase reporter gene (4T1-luc, Perkin-Elmer, Waltham, MA, USA). Approximately 5×106 viable 4T1-luc cells suspended in 100 µl sterile saline containing Matrigel (BD Bioscience, Bedford, MA) were injected into the second left abdominal mammary fat pad of Kunming mice. The tumors were serially monitored using an IVIS Spectrum instrument (Caliper LifeSciences, Inc.) after the injection of 15 mg/kg D-luciferin. Prior to imaging, the mice were anesthetized using the XGI-8 gas anesthesia system (Caliper LifeSciences, Inc), which allowed control over the duration of anesthesia. Oxygen mixed with 2.5% Isoflurane was used for the initial anesthesia, and 0.5% isoflurane in oxrgen was used during imaging. The tumor-bearing mice were randomly divided into three groups (10 mice per group) with the equivalent bioluminescence. PPAN (0.126 mg/kg)，control Pc (0.0187 mg/kg)，saline was injected into the third group via tail vein. After 3 h post injection, mice were exposed to a 680 nm light source for 3 min to a light dosage of 30 J/cm2. Tumor burden was assessed by bioluminescence imaging as increasing bioluminescence.

Stability of PPAN.


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We evaluated the stability of PPAN by comparing the data of DLS sizes analysis, zeta potentials, uv-vis absorption spectra in PBS and DMSO, fluorescence emission spectra in DMSO, and the ROS generation in PBS between a freshly prepared batch of PPAN and an old batch

standing for 60 days. The old sample was exposed to the environmental light with the day-night cycles at room temperature for 60 days. The processes of the determination of the above data were referred to the above relevant descriptions.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXXXX. The supporting information provides the further data of the Experimental Section, synthesis scheme of mono-Pc, the assembly process of PPAN simulated by MD simulation, the raw data of flow cytometry experiments, merged graphics of the transparent and fluorescent fields of ex vivo imaging, and other supplementary data.

AUTHOR INFORMATION Corresponding Authors * J. L.: email: [email protected]. * Z. C.: email: [email protected]. * P. X.: email: [email protected].

ORCID Jinyu Li: 0000-0002-8220-049X Longguang Jiang: 0000-0002-4734-3778 Xueyuan Chen: 0000-0003-0493-839X Mingdong Huang: 0000-0002-1377-6786 Zhuo Chen: 0000-0002-6351-8689 Peng Xu: 0000-0003-3968-7279

Notes



The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation of China, grant numbers: 21708043 (P.X.), 21603033 (J.L.), 81572944 (Z.C.), U1405229 (Z.C.); the CAS/SAFEA International Partnership Program for Creative Research Teams; and the CAS Strategic Priority Research Program (XDA09030307); Fuzhou University Testing Fund of precious apparatus, grant number: 2017T010 (L.J.). The authors gratefully acknowledge the computing time granted by the Fujian Supercomputing Center.

AUTHOR CONTRIBUTIONS M.H., Z.C., and P.X. conceived and designed the project. Y.J. performed the experiments. J.L. carried out the molecular dynamic simulation. Y.J. and P.X. analyzed the data. J.C., P.H., L.J., and X.C. provided technical supports. Z.C. and P.X. finished the writing.

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