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pH Triggered Peptide Self-Assembly for Targeting Imaging and Therapy towards Angiogenesis with Enhanced Signals Yixia Qian, Weizhi Wang, Zihua Wang, Xiangqian Jia, Qiuju Han, Iman Rostami, Yuehua Wang, and Zhiyuan Hu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b00583 • Publication Date (Web): 14 Feb 2018 Downloaded from http://pubs.acs.org on February 14, 2018

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ACS Applied Materials & Interfaces

pH Triggered Peptide Self-Assembly for Targeting Imaging and Therapy towards Angiogenesis with Enhanced Signals Yixia Qian,§,a,b Weizhi Wang,§,*,a Zihua Wang,a Xiangqian Jia,a Qiuju Han,a Iman Rostami, a Yuehua Wang,a Zhiyuan Hu*,a,c,d a. CAS Key Laboratory of Standardization and Measurement for Nanotechnology， CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience，National Center for Nanoscience and Technology of China, Beijing 100190, China. b. University of Chinese Academy of Sciences, Beijing 100049, P. R. China c. Sino-Danish College, University of Chinese Academy of Sciences, Beijing 100049, P. R. China. d. Centre for Neuroscience Research, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian Province, China. Keywords: pH triggered • VEGFR2 • self-assembly • bioimaging• cancer therapy ABSTRACT Mild acidic environment and angiogenesis are two typical characteristics of tumor. The specific response towards both lower pH and angiogenesis may enhance the targeting ability both for drug and diagnostic probe delivery. Herein, we present a kind of dual responding self-assembled nanotransformation materials which are tumor angiogenesis targeting and pH triggered based on amphiphilic conjugation between peptide (STP) and aromatic molecule (TPE). The morphology of the self-assembled peptide-conjugates is responsibly changed from nanoparticles in neutral condition to nanofibers in acidic condition, which “turn on” the in vivo targeting imaging and accelerated the efficient drug delivery and in vivo therapy. Based on the well-controlled nanotransformation both in vitro and in vivo, we envisioned the successful demonstration of the responding materials would open a new avenue on “turn on” targeting imaging diagnostics and specific cancer therapeutics.

1.

Introduction One of the chemical research field pertaining to cancer research have been ACS Paragon Plus Environment

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concentrated on the development of suitable vehicles which can be loaded with anticancer drugs or diagnostic reagents and deliver them specifically towards cancer sites1. Functional nanomaterials are desirable bioactive materials for cancer diagnosis and therapy. Well-controlled self-assembly at biointerfaces is an effective way to construct nanostructures for biocompatible delivery2-4. Among them, amphiphilic molecules consist of a hydrophobic moiety and a hydrophilic moiety are inclined to self-assembled into specific nanostructures5-6, widely applied in biomedical field, including drug delivery, in vivo imaging and therapy7-9. Precisely control over the self-assembly process to adapt the complex biological environment is demanded. Consequently, to develop biocompatible and controllable self-assemble molecules as well as triggered by the physiological environment is crucial. At present, various strategies have been developed to control over the self-assembled structure, including catalyst, pH and light10-13. Peptides with high diversity have been investigated as building blocks for nanostructures14. The sequences and secondary structures of peptides can also impact on the nanostructure formation15-17. Peptide based self-assembly nanostructures, including micelles, tubes, fibres, or ribbons are satisfactory biocompatible carriers18-20. On the other hand, molecules with conjugated π-bond structure could be served as hydrophobic building blocks to design ordered structures via self-assembly, while in some case, these molecules also show sensitive signals. The integrated self-assembly nanostructures formed by the combination of hydrophilic peptides with hydrophobic signal molecules could realize biocompatible molecular self-assembly with controlled nano structures which could be utilized in in vivo tracing, imaging and targeting delivery21-24. Furthermore, the stimuli-responsive draws more attention to study the specific physiological microenvironments25-29. Acidic tumor microenvironment (pH 5.5-6.9) is caused in the process of tumor development, which associates with the tumorigenesis, invasion and metastasis30. In addition, angiogenesis plays a crucial role in the tumor growth. Vascular endothelial growth factor receptor 2 (VEGFR2), as angiogenesis related growth factor receptor secreted in the new formed tumor vessels, is a typical ACS Paragon Plus Environment

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tumor marker31-32. Therefore, angiogenesis targeting is also an efficient way for cancer theragnostic. Above all, developing of angiogenesis targeting and pH triggered materials with enhanced imaging and delivery efficiency is significant. Herein, we reported a pH triggered peptide self-assemble material through the conjugation between hydrophilic peptide and hydrophobic signal molecule, showing self-assembly enhanced signals for targeting imaging, drug delivery and cancer therapy.

STP

(sequence:

SKDEEWHKNNFPLSP),

a

pH

triggered

and

VEGFR2-targeting peptide was severed as hydrophilic building block33-34. It can only bind in the condition of VEGFR2 overexpression and a mild acidic environment. In addition, it formed reversible structures that α-helix in acidic environment while random coil in neutral environment. Hydrophobic signal molecule with the assembly induced enhanced signals (carboxylated tetraphenylethylene, TPE-COOH) was employed as the hydrophobic building block. On one hand, the pH triggered self-assembled materials towards both angiogenesis and low pH showed active targeting effect. On the other hand, the environmental triggered and well-organized nanofibers nanostructure showed higher delivery efficiency than the nanoparticles as well as better therapeutic effect. Therefore, the self-assembled nanofiber materials were endowed better imaging sensitivities and delivery efficiencies even though the tumor was at the early stage.

2.

Materials and Methods

2.1 Materials Doxorubicin hydrochloride was purchased from Hisun Pharmaceutical Corp (Taizhou, Zhejiang, China). Purpurin 18 was purchased from Xianhui Pharmaceutical Technology

Co.,

Ltd

(Shanghai,

China).

9-Fluorenylmethoxycarbonyl

(Fmoc)-protected amino acids, Wang resin was purchased from GL Biochem (China). Trifluoroacetic acid (TFA), thiazolyl blue tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), lysis buffer was from Sigma-Aldrich (USA). Benzophenone was from Sinopharm Chemical Reagent Co.,Ltd. 4-methoxybenzophenone was from Alfa Aesar (Beijing, China). Triisopropylsilane (Tips) was from Acros Organics (USA). ACS Paragon Plus Environment


BCA protein assay kit was from Beyotime Biotechnology (China). PVDF membranes was form Merck Millipore (Germany). VEGFR2 protein was from Sino Biological Inc (Beijing, China). DMEM/high glucose medium and trypsin were purchased from GE Healthcare Life Sciences. The human umbilical vein endothelial cell line HUVEC, the human breast cancer cell line MCF-7 were purchased from Cell Resource Center, Chinese Academy of Medical Sciences (China). All cells were supplemented with 10% fetal bovine serum (FBS) (Gibco) and 100U/mL penicillin and 100U/mL streptomycin (Gibco). Other regents were all from Beijing Chemical Plant (Beijing, China). 6-8 week-old female BALB/c nude mice were purchased from Vital River Laboratory Animal Technology Co., Ltd (Beijing China).All reagents were used as received and the solvents were purified according to the general procedures before used. 2.2 Synthesis and characterization of 2-[4-(1,2,2-triphenyl-1-ethenyl)-phenyloxy] acetic acid (TPE-COOH). TPE-COOH was prepared according to the reported procedures (Figure S1a). Preparation of 1,1,2-Triphenyl-2-(p-methoxyphenyl)-ethene. Benzophenone (1.84 g, 10 mmol), 4-methoxybenzophenone (2.14 g, 10 mmol), and zinc powder (9.65 g, 50 mmol) were added to a three-necked flask, which was then vacuum-evacuated and flushed 3× with dry nitrogen. A 100 mL portion of dry THF was added, and then TiCl4 (3.4 mL, 30 mmol) was added dropwise using a syringe at 0°C. After refluxing overnight, the reaction was quenched by addition of 10% K2CO3 solution (6.9 g in 62.5 mL water). The organic phase was washed 2× with brine and then dried over anhydrous MgSO4. The crude product was filtered, concentrated, and passed through a silica gel column with an eluent of petroleum ether and ethyl acetate (20:1 v/v). The final product was a white solid with a yield of 45%. Preparation of Methyl 2-[4-(1,2,2-Triphenyl-1-ethenyl)-phenyloxy]acetate. A two-necked flask containing 1,1,2-triphenyl-2-(p-methoxyphenyl)ethene (1.82 g, 5 mmol) and dried CH2Cl2(50 mL) was cooled to -20°C, and 1.0mL of BBr3 was slowly added. The mixture was warmed to room temperature, allowed to stand for 4 h, and then poured into saturated aqueous sodium bicarbonate. The organic phase was dried over anhydrous MgSO4, and the crude product obtained after concentration was ACS Paragon Plus Environment

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dissolved in 40 mL of CH3CN. K2CO3 (0.69 g, 5 mmol) and methyl bromoacetate (0.5 mL, 5 mmol) were added, and the mixture was refluxed for 4 h before cooling and filtering. The filtrate was concentrated and purified on a silica gel column with an eluent of petroleum ether and ethyl acetate (10:1 v/v) to give the product (84% yield). Preparation of 2-[4-(1,2,2-Triphenyl-1-ethenyl)phenyloxy]-acetic Acid (TPE -COOH). Methyl 2-[4-(1,2,2-triphenyl-1-ethenyl) phenyloxy]acetate (1.00 g, 2.3 mmol) was dissolved in 75 mL of THF/H2O (v/v = 7:1). LiOH (0.6 g, 25 mmol) was then slowly added, and the mixture was stirred overnight. THF was removed under reduced pressure and the mixture extracted 3× with 30 mL of CH2Cl2. Saturated NH4Cl solution was added to neutralize the aqueous phase, and the pH was adjusted to 1.0 with 1 M HCl before extracting 3× with 40 mL of ethyl acetate. The organic phase was dried over anhydrous MgSO4 and then concentrated to give TPE-COOH (0.76 g, 82% yield). 2.3 Synthesis and characterization of STP and TPE-STP Peptides were synthesized through the Fmoc strategy. Wang Resin was used as the solid phase support. All the synthesis process was carried out in dehydrous DMF in the solid phase peptide synthesis vessels with sieves in it. In the coupling step, the Fmoc-amino acid reagent was dissolved in 0.4 mol/L NMM in DMF and the coupling time was 40 min. In the deprotection step, 20% (v%) piperidine was used to remove the Fmoc group and the deprotection time was 10 min. After elongation, TPE-COOH was added into the DMF solvent with DIEA to activate the carboxyl groups and mix with the peptide. After 24 h, cleavage reagent (95% (v%) TFA, 2.5% (v%) water, 2.5% (v%) Tips) was introduced into the vessel to cleave the resins and SP (side chain protecting group) of each residue (2 h). The crude TPE-STP was analyzed and purified by using a Waters HPLC (High Performance Liquid Chromatography) system (Waters e2695) on a SunFire® C18 column (5 µm, 4.6×250 mm) at a flow rate of 1.0 mL/min. Gradient: 0-25 min, 5-70% acetonitrile containing 0.1% TFA. 2.4 Fluorescence measurements. A stock solution of TPE-STP (1000 µM) was prepared with Milli-Q water. The fluorescence emission spectrum of TPE-STP was measured at room temperature. For ACS Paragon Plus Environment


sensitive and selective detection, TPE-STP was dilute to 10 µM, the VEGFR2 protein and various anions (10 µM) and biomolecules (DNA.RNA 2 µM) were incubated with TPE-STP at 37°C for 60 min. Then the corresponding fluorescence spectrum was recorded by a UV-Vis spectrometer. The excitation wavelength was 340 nm and emission was collected from 400 to 650 nm. 2.5 Prepration of TPE-STP and TPE-STP-DOX TPE-STP nanostructures were prepared by dropping TPE-STP solution into different pH Milli-Q water, then oscillating with a vortex. TPE-STP-DOX were fabricated by dropping TPE-STP solution into Milli-Q water and oscillating as before, then immediately adding an aqueous DOX solution (20% of molar TPE-STP concentration) and oscillating again. 2.6 In vitro confocal fluorescence imaging of HUVEC cells Approximately 1×105 cells/mL were seeded into 35 mm microscopy dishes and cultured overnight for the adhesion of cells. The stock solution of TPE-STP was diluted with DMEM medium to a concentration of 80 µM. After the cells had been washed with cold DMEM medium three times, TPE-STP (200 µL) was added. After incubation for a certain period, the fluorescence images were collected directly using ZEISS LSM710 confocal microscope without further washing of the samples. For drug delivery, HUVEC cells were treated with TPE-STP-DOX (80 µM) in DMEM medium for 1.5 h. Then removed the solution and added 200 µL Lysotracker Green DND-26 (0.05 µM) culturing for a further 30 min. Finally, cells were washed with DMEM three times and detected by confocal microscope. For TPE, the excitation was 405 nm, and the emission filter was 466±10 nm; For DOX detection, the excitation was 514 nm, and the emission filter was 591±10 nm. For Lysotracker detection, the excitation was 488 nm, and the emission filter was 511±10 nm. 2.7 Atomic force microscopy (AFM) images. AFM images were obtained in a scanasyst mode using a scanning probe microscope (Bruker Multimode-8). A stock solution of TPE-STP (1000 µM) was prepared with Milli-Q water. Then it was diluted to 40 µM in water with pH 5.8 and pH 7.4. Samples were prepared onto a freshly cleaved mica surface for 15 min. The ACS Paragon Plus Environment

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AFM data was analyzed using a NanoScopeAnalysis software. 2.8 In vitro and in vivo photoacoustic imaging Potoacoustic molecule purpurin 18 was served as the signal molecule for imaging. P18 was loaded with the TPE-STP. The self-assembled TPE-STP-P18 and TPE-TP-P18 were first studied in vitro at acidic condition. Sample with a final concentration of 100 µM was added in tissue-mimic phantom. The photoacoustic signals and intensity was obtained from a multispectral optoacoustic tomography (MOST 128, iTheraMedical, Germany). The in vivo experiment was performed on the 6-8 week-old female BALB/c nude mice. All animal experiments were performed in accordance with protocols approved by the Committee for Animal Research of Peking University, China. MCF-7 cells were subcutaneously implanted into mice on the right flank. Tumor size was measured periodically using callipers. 200 µL peptide-conjugates were intravenously injected into the tumor-bearing nude mice (400 µM, 4% DMSO in PBS), respectively. Images were taken at 1 h, 3 h, 6 h post-injection and analyzed with a MOST in vivo imaging system. The photoacoustic signal intensity was recorded through mean pixel intensity of the same area of the images. 3D reconstructions were generated from the photoacoustic images by Image J software using continuous tomography with a space interval of 0.2 mm in a range of 2 cm. 2.9 In vivo therapy assays of TPE-STP-DOX, TPE-TP-DOX and free DOX. Female BALB/c nude mice of about 6-8 week-old were purchased from Vital River Laboratory Animal Center (Beijing, China), and kept under specific pathogen-free conditions with free access to standard food and water. All animal experiments were performed in accordance with protocols approved by the Committee for Animal Research of Peking University, China. MCF-7 cells were subcutaneously implanted into mice on the right flank. After the tumors had been allowed to develop to approximately 80-100 mm3, in vivo tumor suppression studies were carried out to examine the toxicity and tumor inhibition efficiency of TPE-STP-DOX, TPE-TP-DOX, free DOX. Mice were injected intravenously with PBS (control group), TPE-STP-DOX, TPE-TP-DOX, free DOX (200 µL) at a dose corresponding to ACS Paragon Plus Environment


5mg/kg of DOX (n=3). Administration was carried out every other day for three times. The weights and tumor sizes were recorded daily at the same time. Tumor sizes were measured by a vernier caliper. Tumor volume was calculated by the formula (L×W2)/2. L is for the longest and W is the shortest in tumor diameters (mm). 2.10 Slicing experiment and detection of the organs To evaluate levels of apoptosis in tumor cells, tumor tissue sections were stained by terminal deoxynucleotidyl transferased dUTP nick end labelling (TUNEL) assay according to the manufacturer's protocol (KeyGEN BioTECH, Nanjing, China). The nucleus was counterstained with DAPI and examined by confocal microscope. Furthermore, the heart, liver, spleen, lung, kidney and tumor were used for histopathology analysis. 3.

Results and Discussions

3.1 Design, synthesis and characterization of the self-assembled TPE-STP. The amphiphilic self-assemble nanostructures were first constructed. Carboxylated tetraphenylethylene (TPE-COOH), an aggregation induced emission (AIE) molecule was selected as the hydrophobic building blocks for self-assembly and also served as the chromophore to realize fluorescence “turn on” towards VEGFR2 protein. TPE-COOH was prepared according to the reported procedures and characterized by HNMR spectrum and mass spectrum (Figure S1)35-36. The TPE-STP was rationally designed following the solid phase peptide synthesis and characterized by HPLC and mass spectrum (Figure S2). Figure 1a showed the self-assembly process of the TPE-STP. The pH triggered peptide STP formed reversible structures that α-helix in acidic environment while random coil in neutral environment. Therefore, TPE-STP also showed the reversible structures during the microenvironmental transformation. Direct evidences were from AFM images. As shown in Figure 1b, the self-assembled morphology of TPE-STP was formed at both pH values (acidic and neutral), it revealed that at neutral pH, TPE-STP behaved as nanoparticle structures with low densities. However, at acidic pH, quite different morphology was obtained. TPE-STP self-assembled into nanofibers in acidic environment with higher densities. The single

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fiber had diameters of 6-8 nm and height of 3.8 nm (Figure S3). These fibers lengths up to several hundred nanometers and some of them had the tendency to combine together. We considered it owning to the intra- and intermolecular interactions within TPE-STP. For another reason, the secondary structure of peptide STP was also crucial in the self-assemble process because of the proved α-helix structure specific in acidic environment

37-38

. The pH change of TPE-STP solution leaded to STP configuration

changed and successively influenced the morphology of self-assembled TPE-STP. Furthermore, in order to check the self-assembly status after the molecule loading, cancer chemotherapy drug doxorubicin (DOX) was loaded on the self-assembled TPE-STP conjugate. As shown in Figure 1c, when drug was loaded in the TPE-STP nanostructure, pH triggered nanofiber structure could be induced with even higher fiber density. It may result from more electrostatic interaction between molecules. As a molecule environment triggered carrier, TPE-STP could still perform the self-assembled ability which was of significance for cellular delivery (Figure 1d) and in vivo tracing (Figure 1e). 3.2 Characterization of the specific “turn on” conjugate and fluorescence imaging of targeting the VEGFR2 protein in living cells. We investigated the performance of the TPE-STP nanostructure in molecular and cellular level. First, fluorescence titration experiments were carried out to check the specific signal “turn on” of self-assembled TPE-STP towards VEGFR2 protein at different conditions. In the absence of VEGFR2 protein, TPE-STP showed low fluorescence intensities. At pH 5.8, with an increasing amount of added VEFGR2 protein (up to15 µg), the fluorescence intensity was gradually enhanced (Figure 2a). The signal ratio between saturation intensity and initial intensity (S/I) was more than 4. Analysis of the titration data revealed that the emission intensity increased linearly (R=0.99) (Figure S4). However, the fluorescence intensity was kept at a relative low level at pH 7.4 (Figure 2b and S5) which showed the S/I less than 1.5. The fact that the fluorescence increased upon addition of VEGFR2 protein was owing to the specific binding of STP towards VEGFR2 protein at acidic environment only, which ACS Paragon Plus Environment


inhibited the internal rotations of the TPE framework and initiated the aggregation of self-assembled TPE-STP simultaneously. To further confirm the high specificity and good selectivity of self-assembled TPE-STP, an analogue of VEGFR2 aminopeptidase N (APN)

39-40

and other important biomolecules such as glucose, ATP, PO43-, DNA

and RNA were tested. As illustrated in Figure 2c and S6, TPE-STP showed a high selectivity towards VEGFR2 protein. What’s more, only at mild acidic condition, the fluorescence intensity increased (Figure S7). It is indicated that the self-assembly of TPE-STP kept their own properties, showing dual responding specificity towards VEGFR2 protein. The self-assembled TPE-STP was further tested on living cells. Human umbilical vein endothelial cell (HUVEC) was chosen as VEGFR2 overexpression cell model41-42 and verified by western blot assay (Figure S8). HUVEC was incubated with TPE-STP and monitored by confocal laser-scanning microscopy (CLSM). As shown in Figure 2d, without any washing step, high-contrast staining of the cell membrane was achieved in acidic condition. However, at neutral environment, little fluorescence was detected. Only if the mild acidic condition and the VEGFR2 were both in presence, the self-assembled TPE-STP was responded and the fluorescence would be switched on. In addition, we carried out a competitive assay. HUVEC was pre-treated with STP for 30 min before incubation with TPE-STP. There was still no fluorescence. We considered it owning to the preoccupancy of the extracellular VEGFR2 sites by STP, which blocked the binding sites of self-assembled TPE-STP towards VEGFR2. These results confirmed the specific signal “turn on” of the self-assembled TPE-STP towards VEGFR2 in living cells only by the response of both pH and the angiogenesis marker. The signal was more obvious due to the nanofiber formation. Time-course assay was also carried out in acidic condition to monitor the behavior of TPE-STP into HUVEC cells (Figure 2e). Along with the incubation time, a brighter fluorescence was detected and after 90 min incubation, a great intensified fluorescence appeared in the cytoplasm. It was indicated that the self-assembled nanofiber structures could penetrate into cytoplasm with the nanofiber formation.


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3.3 Fluorescence imaging of the responding self-indicating targeting and drug delivery system in a low-pH environment. The self-assembled TPE-STP was applied as a targeting drug carrier with “turn on” signals. We continued to construct a responding self-assembly drug delivery system (TPE-STP-DOX) and investigated the targeted drug delivery efficiency of TPE-STP towards living cells in acidic environment. TPE-STP-DOX was incubated with HUVEC cells for 1.5 h in acidic condition. None-load TPE-STP and free DOX were served as control. As shown in Figure 3, the signal of TPE-STP (Figure 3a and Figure 3b blue channel) was only visible in the cytoplasm, suggesting the nanofiber carrier didn’t come into nuclei. In the TPE-STP-DOX group, most fluorescence of DOX (Figure 3a red channel) was shown in nuclei. In the free DOX control group, DOX was still much remained in cytoplasm, revealing lower transport efficiency. So, we draw a conclusion that the self-assembled TPE-STP delivery much more DOX into the nuclei than free DOX itself. Notably, in the DF (dark field) merge images, there were interesting regions where the blue and red fluorescence signals were overlaid, showing violet signals. They were supposed as lysosome where the drug released. To confirm our estimation, we used the Lysotracker Green to stain the lysosomes and found the green fluorescence signals were co-localized with the violet ones. To further confirm the final localization of DOX, we increased the incubated time up to 2.5 h and found that almost all the red signals were visible in the nuclei (Figure S9). We also changed the sensitive peptide STP with another none-sensitive peptide TP (sequence: TIDHEWKKTSFPLSF) (Figure S10). The DOX delivery efficiency decreased greatly (Figure S11). These results indicated that the self-assembled TPE-STP-DOX can recognize the cell membrane receptor selectively, penetrate into the cytoplasm and release DOX quickly. Since the effective binding site of DOX was DNA in the nuclei, it showed enhanced drug delivery ability by the TEP-STP carrier while performed the fluorescent indicating ability simultaneously. Furthermore, the drug loading efficiency (DLE) and drug loading content (DLC) of TPE-STP-DOX were evaluated according to the DOX fluorescence intensity standard curve (Figure



S12). DLE of TPE-STP-DOX was calculated as 89.7%, which suggesting a high drug loading efficiency while that of TPE-TP-DOX was 86.6%. In addition, DOX releasing behaviors of TPE-STP-DOX and control one (TPE-TP-DOX) were investigated in vitro in the dialysis model at 37°C (Figure S13). It’s revealed similar drug release behaviors. Furthermore, cytotoxicity of the TPE-STP towards HUVEC cells was also evaluated by MTT (Figure S14). It revealed that the material was highly biocompatible. TPE-STP-DOX was more effective in inhibiting the cell proliferation than that the free DOX at the same concentration. These may result from higher penetrating ability of the self-assembled TPE-STP. What’s more, the pH sensitive effect on cell cytotoxicity was evaluated at pH 5.8 and pH 7.4. The result showed that at acidic environment, the TPE-STP-DOX performed a higher cell inhibited effect (Figure S15). Cellular uptake of DOX was measured by flow cytometry (Figure S16). The cells treated with TPE-STP-DOX contained more DOX than that of free DOX at same incubation time. The phenomenon was more obvious when the incubation time reached 2.5 hours. 3.4 Signal molecule delivery by the self-assembly nanofiber for detection tumor in an early stage Encouraged by effective drug delivery in cellular level, in vivo deliver assay was achieved. In order to realize a diagnosis-therapy combined integrated delivery system, sensitive signal molecules and drug were both loaded in the TPE-STP nanostructure. First, to achieve a dual responding in vivo imaging, a photoacoustic molecule Purpurin 18 (P18) was loaded in TPE-STP to prepare TPE-STP-P18. The in vivo experiment was performed on a MCF-7 cell xenografted mouse model with VEGFR2 high expression at tumor site

43-44

(Figure 8). The tumor was in an early stage and the

average diameter was around 2 mm. Photoacoustic imaging of tumor (transverse section) was carried out on a multispectral optoacoustic tomography (MOST, Figure 4a). Similarly, none pH sensitive peptide TP was chosen as control (TPE-TP-P18). Self-assembled TPE-STP-P18 was first studied in tissue-mimic phantoms (Figure 4b). The photoacoustic signal of TPE-STP-P18 was 1.5 times higher than control one.


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Then we move on to the in vivo checking. TPE-STP-P18 was injected via tail vein. Time-dependent photoacoustic images and photoacoustic signal intensity in tumors treated with TPE-STP-P18 and control ones (TPE-TP-P18) were shown in Figure 4d and e. The photoacoustic signal of TPE-STP-P18 in tumor was intensively increased at 6 h post-injection, in which we inferred that the self-assembled was triggered by the acidic tumor microenvironment and the VEGFR2 receptor. The signal ratio between tumor site and normal tissue was up to ~8 (ratio=8000/1000=8). Using continuous tomography, 3D reconstruction of the tumor site after 6 h post-intravenous injection was performed (Figure 4c). For TPE-TP-P18, it also showed photoacoustic signals at tumor site at 6 h, but the intensity was weak and barely visible. We also carried out a control experiment to analyze the real-time biodistribution of the nanostructure in a non-tumor bearing mice, as shown in Figure S17, there is no obvious aggregation in the tissues or organs. Therefore, we speculated that with the pH triggered and VEGFR2 targeting peptide, the self-assembly morphology of TPE-STP-P18 was responded to the tumor acidic microenvironment and experienced the nanofiber transformation, which obviously improved the accumulation of signal molecules in tumor site. 3.5 In vivo tumor therapy by the nanostructure based drug delivery The ultimate purpose of developing the dual responding self-assembly material was to realize an efficient diagnosis and therapy. Therefore, a tumor inhibition study in vivo was performed. The MCF-7 tumor-bearing mice (80-100 mm3) were randomly divided into four groups (n=3). Each group was treated intravenously with TPE-STP-DOX, TPE-TP-DOX, Free DOX and PBS, respectively. The cure progress was terminated after 17 days. Administration was carried out every other day. After the whole assay, tumors were excised. As shown in Figure 5a, the tumor growth of the TPE-STP-DOX treating group was obviously inhibited when compared with PBS treating group. While TPE-TP-DOX and free DOX group tumor growth was inhibited a little. These results indicated both the acidic tumor environment and the tumor vessel marker VEGFR2 have triggered the materials. The tumor size and body



weights were also recorded daily during the therapeutic process (Figure 5b and c). The body weight of TPE-STP-DOX treating group changed little, indicating low toxicity. After the treatment, the tumors and main organs were exteriorized from the mice. Histological examination of the tumors was evaluated. As shown in Figure 5d, hematoxylin-eosin (H&E) staining of the tumor demonstrated TPE-STP-DOX group exhibited large damage of tumor tissues. Additionally, the apoptosis of tumor cells was also detected by TUNEL assay (Figure 5e). The cell apoptosis of TPE-STP-DOX group was more obvious than control groups. Other main organs were also analysed by H&E staining (Figure S18). The experiment groups showed no apparent morphological difference among the therapeutic groups in heart, liver, spleen, lung or kidney. Of all the results implied that TPE-STP-DOX realized high efficiency at tumor sites and would not cause serious harm to other nontargeted sites, which expressed a satisfactory therapeutic effect. 4.

Conclusions In summary, dual responding self-assembly materials were developed by

peptide-conjugates, showing pH triggered and angiogenesis targeting properties and endow nanotranformation to well-organized nanofibers in acidic environment. TPESTP nanostructure showed fluorescence “turn on” with high sensitivity and selectivity towards VEGFR2 protein at acidic environment. Self-assembled TPE-STP was then applied in delivery system for self-indicating drug release in cellular level. Furthermore, as a dual responding nanofiber carrier, the self-assembled TPE-STP could enhance the in vivo delivery efficiency towards tumor. Photoacoustic molecule P18 loaded TPE-STP showed specific high signal even in an early stage of tumor. Furthermore, drug loaded nanofiber could enhance the antitumor effect. We envisioned the physiological stimuli-responding in living systems provided potential applications in clinical use.

Supporting Information Figures showing synthesis and characterization of TPE-COOH and TPE-STP; western ACS Paragon Plus Environment

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blot assay of the HUVEC cell and MCF-7 cell lysate towards the VEGFR2 antibody; cytotoxicity of TPE-STP, free DOX and TPE-STP-DOX towards HUVEC cells; flow cytometry quantification of cellular of free DOX and TPE-STP-DOX; detailed TPE-STP-DOX spatiotemporal distribution in HUVEC cells; AFM height images of TPE-STP; in vitro release of DOX from TPE-STP-DOX, TPE-TP-DOX. Photoacoustic images and photoacoustic signal intensity distribution in transverse sections of non-tumor bearing mice up to 6 h after injection with TPE-STP-P18. H&E staining analysis of paraffin section about mice organs. Corresponding Author * E-mail: [email protected], [email protected]. Author Contributions Yixia Qian and Weizhi Wang contributed equally to this work. Notes The authors have declared that no competing interest exists.

Acknowledgements We acknowledge funding from Beijing Natural Science Foundation (L172035 and 2172056), National Natural Science Foundation of China (21775031), Beijing Talents Fund (2015000021223ZK36), Key Research Program of the Chinese Academy of Sciences (KFZD-SW-210), Beijing Municipal Science and Technology Project (Z171100002017013) and “Strategic Priority Research Program” of Chinese Academy of Sciences (XDA09040300). Abbreviations TPE-COOH: 2-[4-(1,2,2-triphenyl-1-ethenyl)- phenyloxy] acetic acid; P18: Purpurin 18; VEGFR2: Vascular endothelial growth factor receptor 2; AFM: Atomic force microscopy; DOX: Doxorubicin; HUVEC: Human umbilical vein endothelial cell.



Figure 1. (a) Peptide (STP)-TPE conjugation and chemical structures of STP, TPE-COOH. The self-assembly and transformation of STP-TPE conjugated by acidic/angiogenesis response. AFM height images of TPE-STP at acidic and neutral environment before (b) and after (c) drug loaded. (d) TPE-STP-DOX nanoparticles were switched to TPE-STP-DOX nanofibers in acidic environment and activated by the VEGFR2 protein. DOX was delivered from lysosomes to nuclei. (e) Tumor acidic-response imaging of TPE-STP-P18, it enhances enhanced the photoacoustic signals.


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Figure 2. VEGFR2 detection with TPE-STP. Fluorescence titration profile of TPE-STP (10 µM) with an increasing amount of VEGFR2 at pH 5.8 (a) and pH 7.4 (b). Selective detection of VEGFR2 with TPE-STP. Fluorescence spectra of TPE-STP (10µM) upon addition of various anions and biomolecules at pH 5.8 (c). (d) Fluorescence images of HUVEC stained with TPE-STP at pH 5.8 and pH 7.4 as well as images of TPE-STP on HUVEC pre-treated with STP. (e) TPE-STP staining along with different time points.



Figure 3. Confocal images of the spatial distributions of (a) TPE-STP-DOX (b)TPE-STP and (c) free DOX in HUVEC cells. Lysosomes were indicated by Lysotracker Green. HUVEC cells were incubating with TPE-STP-DOX (80 µM), TPE-STP (80 µM) and DOX (16 µM) for 1.5 h.


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Figure 4. Photoacoustic images by nanostructure loaded P18. (a) transverse section view of the mouse. (b) tissue-mimic phantoms of the two nanostructure-loaded P18. (c) a typical 3D re-construction of the tumor site in photoacoustic imaging. (d) photoacoustic signal intensity distribution in transverse sections of mice up to 6 h after injection with TPE-STP-P18 (200 µL, 4.0× 10-4 M) (white ellipse is tumor site). (e) photoacoustic signal intensity distribution in transverse sections of mice up to 6 h after injection with TPE-TP-P18 (200 µL, 4.0× 10-4 M) (white ellipse is tumor site).



Figure 5. (a) Excised tumor photograph after the cure process. (b) Tumor size changing curves during the cure process. (c) Mice body weight changing curves during the cure process. Analysis of the tumor slices with different treatment with H&E (d) and TUNEL (e) immunofluorescence staining, respectively.


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Graphical Abstract



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