One-Dimensional Supramolecular Nanoplatforms for Theranostics

Sep 2, 2016 - Versatile, supramolecular nanoplatform for personalized needs, particularly–theranostics, was fabricated by coassembly of peptide amph...
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1D Supramolecular Nanoplatforms for Theranostics Based on Co-Assembly of Peptide Amphiphiles Inhye Kim, Eun Hee Han, Jooyeon Ryu, Jin-Young Min, Hyungju Ahn, Young-Ho Chung, and Eunji Lee Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00966 • Publication Date (Web): 02 Sep 2016 Downloaded from http://pubs.acs.org on September 5, 2016

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1D Supramolecular Nanoplatforms for Theranostics Based on Co-Assembly of Peptide Amphiphiles Inhye Kim,† Eun Hee Han,‡ Jooyeon Ryu,† Jin-Young Min,†,‡ Hyungju Ahn,§ Young-Ho Chung,‡ and Eunji Lee*,† †

Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305764, Republic of Korea ‡

Division of Life Science, Korea Basic Science Institute, Daejeon 305-806, Republic of Korea

§

Department of Life Science & Chemical Materials, Pohang Accelerator Laboratory, POSTECH, Pohang 790-834, Republic of Korea Abstract

We report a simple and facile strategy for the preparation of multifunctional nanoparticles with programmable properties using self-assembly of precisely designed block amphiphiles in an aqueous solution-state. Versatile, supramolecular nanoplatform for personalized needs, particularly – theranostics, was fabricated by co-assembly of peptide amphiphiles (PAs) in aqueous solution, replacing time-consuming and inaccessible chemical synthesis. Fibrils, driven by the assembly of hydrophobic βsheet–forming peptide block, were utilized as a nanotemplate for drug loading within their robust core. PAs were tagged with octreotide [somatostatin (SST) analogue] for tumor-targeting or were conjugated with paramagnetic metal ion (Gd3+)-chelating 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for magnetic resonance (MR) imaging. The two PA types were co-assembled to integrate each PA function into original fibrillar nanotemplates. The adoption of a bulky target-specific cyclic octreotide and β-sheet–forming peptide with enhanced hydrophobicity led to a morphological transition from conventional fibrils to helical fibrils. The resulting one-dimensional nanoaggregates allowed the ACS Paragon Plus Environment

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successful intracellular delivery of doxorubicin (DOX) to MCF-7 cancer cells overexpressing SST receptor (SSTR) and MR imaging by enabling high longitudinal (T1) relaxivity of water protons. Correlation between the structural nature of fibrils formed by PA co-assembly and contrast efficacy was elucidated. The co-assembly of PAs with desirable functions may thus be a useful strategy for the generation of tailor-made biocompatible nanomaterials.

Keywords: co-assembly, peptide amphiphile, drug delivery, magnetic resonance imaging, theranostics, helical nanofibrils

1. Introduction Supramolecular self-assembly of rationally designed peptide amphiphiles (PAs) is highly important as it can be applied to a variety of biomedical applications, such as regenerative medicine,1,2 tissue engineering,3-5 and drug delivery systems,6-8 by adopting functional building blocks into amphiphilic molecules. Among the various nanostructures formed by PAs, one-dimensional (1D) fibrillar aggregates that encapsulate water-insoluble payloads within a hydrophobic core are extremely promising for efficient drug delivery, compared with spherical carriers. This is because of their enhanced drug loading capacity and long circulation time in the bloodstream.9,10 Meanwhile, β-sheet formation of peptides has been recognized as one of the main driving forces for the construction of 1D self-assembled nanostructures in water.11 Incorporation of β-sheet–forming amino acids into a hydrophobic block of PA endows the resulting aqueous nanoaggregates with enhanced stability, compared with those formed by amphiphiles with amorphous alkyl chains, and with good biocompatibility because of their intrinsic biological origin.11 Recently, considerable efforts have been devoted to the preparation of functional nanoparticles (NPs) that would be applicable in such biomedical fields as diagnostics, chemotherapeutics, and drug delivery.12-15 Specifically, theranostic NPs with a dual, therapy and diagnostic, function are in high demand for optimized, personalized treatments of disease. To date, such agents have been mainly ACS Paragon Plus Environment

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prepared using inorganic NPs with good optoelectronic and magnetic properties, through attachment of biofunctional ligands.16-19 Regardless, an emerging exists need for a bio-friendly fabrication of customized theranostic agents using facile synthetic procedures. Octreotide, as a synthetic eight amino acid somatostatin (SST) analogue for tracking tumor cells, is well-known for its high resistance to enzymatic degradation, resulting in longer half-life in the plasma compared with the degradation time of SST.20,21 The octreotide analogue is mainly recognized by SST receptor (SSTR) subtype 2 and, to a lesser extent, by subtype 5, both of which are overexpressed by tumor cells. Binding is followed by internalization of receptor-mediated endocytosis.21 Meanwhile, among T1 contrast agents for magnetic resonance imaging (MRI),22 paramagnetic gadolinium ion (Gd3+) is especially attractive because of its high magnetic moment as well as relaxation efficiency.23 This led to a development of commercially available agents, such as Magnevist, Dotarem, and Gadovist. MRI using such contrast medium has been used as a noninvasive diagnostic tool with high spatial resolution in clinical radiology, allowing cellular fate-mapping of living tissue without exposing the subjects to unnecessary ionizing radiation.24,25 However, Gd3+-chelating T1 contrast agents are based on small molecules that have several disadvantages: short half-life and quick extravasation from the vessels.22 In vivo studies using these agents require significant contrast over long periods of time. Since enhanced contrast can be achieved by increasing the molecular weight of an MRI agent, the utility of Gd3+functionalized polymers, PAs, dendrimers, liposomes, and micelles has been explored.26 Therefore, we anticipated that 1D nanofibrils (NFs) based on the self-assembly of PAs with a simultaneous attachment of octreotide and Gd3+-chelating ligand would provide a nanoscaffold suitable for a biocompatible theranostic agent, with a prolonged blood-circulation time, leading to enhanced MR efficacy. However, synthetic concerns had to be addressed. Indeed, synthesis of such PAs resulted in low yields and compounds that were difficult to purify (Figures S1 and S2). In this context, we proposed that a coassembly of PA analogues with specific bioactivities would comprise a facile approach for the preparation of theranostic nanoagents, replacing time-consuming and inaccessible chemical synthesis. ACS Paragon Plus Environment

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Figure 1. Schematic illustration of 1D supramolecular nanoplatform prepared by co-assembly of functional PAs for theranostic purposes. Following the above reasoning, an original fibrillar template for the attachment of therapeutic and diagnostic moieties was achieved by adopting peptide building blocks with a propensity for β-sheet formation (Figure 1).27 Hydrophobicity of the β-sheet–forming peptide within the fibrillar core was controlled, to obtain robust nanoaggregates in aqueous solution. Specific tumor cell-targeting and imaging properties were introduced by each addition of tumor-homing octreotide and 1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for Gd3+-chelation to the outermost of the fibril nanotemplates. Consequently, PAs labelled with octreotide self-assembled into NFs in aqueous solution, and successfully showed target-specific intracellular delivery of hydrophobic anticancer drug doxorubicin (DOX) to MCF-7 cells (human breast adenocarcinoma cells) overexpressing SSTR. Remarkably, co-assembly of PAs containing octreotides and PAs with Gd3+-DOTA chelates resulted in a successful formation of NFs that can be potentially used as MRI nanoprobes. Large amounts of Gd3+ accumulated at the NF surface. High longitudinal (T1) relaxivity of water protons was generated in the co-assembled supramolecular PAs. Thus, the developed fibrillar nanoaggregates can act as an “all-inone” workstation for simultaneous diagnosis and treatment of specific tumor cells. This would provide a ACS Paragon Plus Environment

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useful strategy for developing multifunctional nanomaterials through easy addition of desired capacities to a single nanoplatform.28 2. Experimental section 2.1. Materials Rink Amide MBHA (100-200 mesh) and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) from Merck were used as received. Fmoc-Lys(Boc)-OH, Fmoc-Phe-OH and Fmoc-Thr-(tBu)-OH were purchased from Merk and Fmoc-ᴅ-Phe-OH, Fmoc-ᴅ-Trp(Boc)-OH, Fmoc-Cha-OH, Fmoc-Cys(Acm)-OH and gadolinium(lll) chloride (GdCl3) anhydrous powder were obtained from Sigma-Aldrich and tri-tert-butyl 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (DOTA) was purchased from Tokyo Chemical Industry Co., Ltd. N,N-dimethylformamide (DMF, 99.5%) from Fisher and dicholoromethane (DCM, 99.5%), N-methylpyrrolidone (NMP, 99.7%), piperidine (99%) from Daejung Chemicals and N-ethyldiisopropylamine (DIPEA, 99%) from Merck were used as received. Trifluoroacetic acid (TFA, 99%) from Sigma-Aldrich, methyl phenyl sulfide (99%) and 1,2-ethanedithiol (98%) from Merck were used as received. The N-(Fmoc-8-amino-3,6dioxaoctyl)succinamic acids (Fmoc-PEG2-Suc-OH) were synthesized according to the experimental procedure reported in literature (Figure S1a).29 HeLa cells were obtained from American Type Culture Collection (Manassas, VA, USA) and MCF-7 and CHO-K1 cells (Chinese hamster ovary-K1 cells) were purchased from American Type Culture Collection (Rockville, MD, USA) and from the Korean cell line bank (Seoul, Republic of Korea), respectively. Doxorubicin hydrochloride (DOX·HCl, Boryung Pharmaceutical Co., Ltd., Seoul, Republic of Korea) was desalinated before use. 2.2. Synthesis of C-Cha-DOTA C-Cha-DOTA was synthesized on Rink Amide MBHA resin by conventional solid phase peptide synthesis procedure by using CEM Focused MicrowaveTM Synthesis System, Discover (Figure S1b).30 After the resin was washed with DCM, it was swollen in 1:1 mixed solvent of DMF and DCM for over ACS Paragon Plus Environment

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30 min in shaking incubator. Fmoc deprotection was performed with 20% piperidine in DMF at 75 °C for 3 min. Amino acids (5 equiv.) were sequentially coupled to the N-terminus of peptide on resin using HBTU (2.7 equiv.) and DIPEA (5 equiv.) as coupling agents. In order to introduce DOTA to C-Cha, Dde-Lys(Fmoc)-OH was chosen as a linker where two protecting groups (Fmoc- and Dde-) can be selectively removed. After removal of Fmoc group with 20% piperidine in DMF, DOTA (3 equiv.) was coupled to ε-NH2 of Lys. And then, Dde group was removed with a mixture of DMF:hydrazine = 98:2 at 30 °C at 200 rpm. Elongation of C-Cha-DOTA was accomplished by sequential coupling of each amino acid. After completion of the chain assembly, the resin was treated with a cleavage solution (TFA:1,2-ethanedithiol:thioanisole = 95:2.5:2.5) for over 2 h and the mixture solution was triturated with tert-butyl methyl ether. The final product was purified by reverse phase HPLC on C-18 column using linear gradient of water (0.1 wt% TFA) and acetonitrile (0.1 wt% TFA). The molecular weight of product was confirmed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry (Figure S2). 2.3. Synthesis of N-Phe, A-Phe, and A-Cha N-Phe and PAs having linear octapeptide (A-Phe and A-Cha) were synthesized as the same Fmoc chemistry procedure (Figure S3). After completion of the chain assembly, the resin was treated with a cleavage solution (TFA:1,2-ethanedithiol:thioanisole = 95:2.5:2.5) for over 2 h and the mixture solution was triturated with tert-butyl methyl ether. The resulting products were purified by reverse phase HPLC on C-18 column (SUPELCO, Discovery® BIO Wide Pore C-18, 5 µm, 10 x 250 mm) using linear gradient of water (0.1 wt% TFA) and acetonitrile (0.1 wt% TFA) (Figure S4). The molecular weight of products was confirmed by MALDI-TOF/TOF mass spectrometry (Figure S2). 2.4. Synthesis of C-Phe and C-Cha C-Phe and C-Cha were synthesized as the same procedure described above (Figure S3). For deprotection of the protecting group of cysteine, S-acetamidomethyl (Acm), followed by formation of ACS Paragon Plus Environment

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disulfide (S-S) bond, 1.2 equiv. of thallium trifluoroacetate (Tl(CF3CO2)3) was added to a suspension of the peptidyl resin in DMF:anisole = 19:1 and stirred for 18 h at 0 °C and then the cleavage cocktail (TFA:1,2-ethanedithiol:thioanisole = 95:2.5:2.5) was added to cleave all the protecting groups of amino acids. The final products were purified by reverse phase HPLC on C-18 column using linear gradient of water (0.1 wt% TFA) and acetonitrile (0.1 wt% TFA) (Figure S4). The molecular weight of products was confirmed by MALDI-TOF/TOF mass spectrometry (Figure S2). 2.5. Synthesis of Cha-DOTA DOTA-conjugated PA (Cha-DOTA) was synthesized as the same procedure described above (Figure S3). The final products were purified by reverse phase HPLC on C-18 column using linear gradient of water (0.1 wt% TFA) and acetonitrile (0.1 wt% TFA) (Figure S4). The column eluents were monitored by UV absorbance at 230 and 254 nm. The molecular weight of Cha-DOTA was verified using MALDI-TOF/TOF mass spectrometry (Figure S2). 2.6. Synthesis of fluorescein isothiocyanate (FITC)-labelled octreotide FITC was dissolved in DMF at 10 mg mL-1. 88.7 µL of 22.7 mм of FITC from stock solution was taken, and then put into glass vial. 100 µL of 1.91 mм octreotide was added and stirred at 600 rpm overnight in dark. The resulting FITC-labelled octreotide was purified by HPLC and confirmed by using MALDITOF/TOF mass spectrometry. Calculated [M+2H]2+: 705 (Figure S5).31 2.7. Transmission electron microscopy (TEM) A drop of each sample in aqueous solution was placed on a formvar/carbon-coated copper grid and allowed to evaporate under ambient conditions. When sample was stained, a drop of uranyl acetate solution (2 wt%) was placed onto the surface of the sample-loaded grid. The sample deposited about 1 min at least, and excess solution was wicked off by filter paper. The specimen was observed with a

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JEOL-JEM-3011 HR operating at 300 kV and JEM-1400 operating at 120 kV. The data were analyzed with Gatan Digital Micrograph and Simple Measure programs. 2.8. Circular dichroism (CD) measurement CD spectra were measured using a JASCO J-815 spectro-polarimeter. Spectra were monitored from 190 nm to 260 nm using a 1.0 mm light path length cuvette, and scans were repeated three times and averaged. Molar ellipticity was calculated per amino acid residue. 2.9. Wide-angle X-ray scattering (WAXS) measurement WAXS measurement was performed on PLS-II 9A beamline in the Pohang Accelerator Laboratory (PAL, Republic of Korea). The X-rays generated from the in-vacuum undulator (IVU) were monochromated by Si(111) double crystals and were focused on the detector position using K-B-type mirror system. X-rays with a wavelength of 11.065 Å were used. The thin films were as-cast from a solution of NFs on silicon substrates. Before casting of each sample solution, the Si-wafer was cleaned by sonication in chloroform, acetone, and isopropanol, respectively, for 20 min each. The cleaned substrate was transferred in piranha solution and heated for 1 h at 100 °C. 2.10. Preparation of Nile-red encapsulated NFs Nile red (2 mol %, 10 µм) dissolved in acetone (100 µL) in vial was added to 1 mL of PA solution and then sonicated. The acetone was evaporated by opening the vial cap overnight or till the volume of the solution became about 950-1000 µL. The solution was then lyophilized to remove any remained acetone. The dried residues were re-dissolved in 0.1 м NaCl solution. The absorption spectra obtained from UV-Vis spectroscopy (UV-1800, Shimadzu) at 10 mm of path length in the range of 200-900 nm. Fluorescence spectroscopy (PerkinElmer LS-55) of pure NFs solution and Nile red-encapsulated NFs solution were compared (10 mm of path length, λex = 550 nm). The critical micellar concentration (CMC) values of PAs in water were calculated by serial dilution from 1000 to 1 µм. ACS Paragon Plus Environment

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2.11. Preparation of DOX encapsulated NFs The DOX·HCl was first dissolved in deionized water, followed by addition of triethylamine (TEA) with the molar ratio of DOX:TEA = 1:3 to afford free DOX solution. The mixture was vigorously stirred overnight under dark condition. The prepared hydrophobic DOX (2 mм) was introduced into the PA solution in acetone with the molar ratio of DOX to PA 3:1 and then sonicated. The solvent was evaporated under reduced pressure and then distilled water was added to afford 1 mм of NF solution. The aqueous solution was put into a dialysis tube (molecular weight cut-off 14000 Da) and subjected to dialysis for 10 h under dark with stirring. The distilled water was replaced every 2 h to remove unloaded drug. Samples were lyophilized and reconstituted in distilled water for drug-loading ability test with spectroscopic analysis or in PBS buffer solution (pH 7.4) for in vitro cellular uptake study. To determine the drug loading content (DLC), the lyophilized DOX-loaded NFs were treated with chloroform and then sonicated for at least 30 min. DOX was collected by centrifugation, and then quantitatively analyzed by UV-Vis spectrometer. The amount of DOX was calculated by UV absorbance at 488 nm using a standard calibration curve. A calibration curve was obtained from DOX in chloroform solution with various concentration by measuring absorbance at λex = 488 nm. DLC and drug loading efficiency (DLE) were calculated according to the following formula, respectively: DLC (wt%) = (weight of loaded DOX/total weight of NFs) x 100 DLE (wt%) = (weight of loaded DOX/weight of DOX in feed) x 100 2.12. In vitro DOX release study The DOX-release from the DOX-loaded NFs was conducted in dialysis tube (in 1 mL), which was suspended in 20 mL of sodium acetate buffer (50 µм, pH 5.5) containing 10% dimethyl sulfoxide (DMSO) at 37 °C with constant shaking at 100 rpm. At certain time intervals, 1 mL of medium was emptied and replenished with the same volume of fresh medium. The amount of release DOX was ACS Paragon Plus Environment

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measured by fluorescence spectrometer (λex = 488 nm) and the cumulative release curve of DOX was obtained. 2.13. Cell culture MCF-7 cells were grown in Dulbecco's Modified Eagle Medium (DMEM, HyClone, Logan, Utah, USA)

containing

10%

fetal

bovine

serum

(FBS,

HyClone,

Logan,

Utah,

USA),

1%

penicillin/streptomycin (P/S, HyClone). CHO-K1 cells were grown in Kaighn's modified F-12K nutrient mixture (F-12K, Gibco–Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS, HyClone, Logan, Utah, USA), 1% penicillin/streptomycin (P/S, HyClone). All cell lines were routinely cultured at 37 °C in humidified atmosphere with 5% CO2. 2.14. Cell viability and cytotoxicity assay Cells were seeded in 96-well plates (100 µL per well) and treated with various concentrations of NFs for desired time. Cell viability and cytotoxicity were measured by cell counting kit-8 (CCK-8, Dojindo, Japan) assay according to the guidelines recommended. 10 µL of CCK-8 was added into each well and incubated with cells for 1-4 h. The absorbance was measured on a microplate reader (Spectra Max M5, Molecular Device Co.) at 450 nm. Cell viability was obtained by comparing the absorbance of treated cells to that of control cells. 2.15. Analysis of cellular uptake by confocal laser scanning microscopy (CLSM) imaging To demonstrate cellular uptake of NFs, cells were seeded in confocal slide at 40,000 cells/well in complete cell culture media. Cell imaging plates were acquired from Ibidi GmbH (Ibidi, Munich, Germany). After treatment with FITC-labelled octreotide, Nile red and DOX-loaded NFs at indicated concentrations for desired time, cellular images of intracellular localization were obtained by using an inverted CLSM installed at the Korea Basic Science Institute (Daejeon, Republic of Korea). Images were obtained with the ZEN2009 software (Carl Zeiss). To stain the nucleus, cells were fixed with 4% ACS Paragon Plus Environment

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paraformaldehyde for 5 min, rinsed 3 times with PBS and then incubated for 10 min with DAPI solution. Cells were mounted in polyvinylalcohol mounting medium with DABCO® (Sigma). 2.16. Flow cytometry analysis Cells were seeded in 12-well plates and treated with NFs for 2 h. After that, medium was removed and cells were harvested by trypsin and then fixed by 4% paraformaldehyde and washed several times with PBS. Cells were analyzed by using flow cytometer (MoFlo Astrios, Beckman Coulter) installed at the Korea Basic Science Institute (Daejeon, Republic of Korea). Laser excitation and emission band pass wavelengths were 488 nm and 576 nm, respectively. The results are reported as the median of the distribution of cell fluorescence intensity obtained by analyzing 10,000 cells in the gate. The results are analyzed by Summit Software (Version 6.0, Beckman Coulter). Values of the internalization score, mean fluorescence intensity and mean side scatter intensity were calculated for at least 5000 cells per sample. 2.17. Preparation of Gd3+-complexes The mixtures of Cha-DOTA and C-Cha were dissolved in deionized water to give a solution with concentration of 0.1 mм. GdCl3 (10-50 equiv.) was added to the NF solution at room temperature. The reaction solution was stirred for 24 h. In order to remove the free Gd3+ ions, the solution was centrifuged twice. The product was characterized using MALDI-TOF/TOF mass spectrometry. Calculated [M+Gd+2H]2+: 1072. 2.18. Contents of paramagnetic Gd3+ complexed to the co-assembled NFs Samples for analysis were prepared by taking 200 µL from the stock solution used for relaxivity and T1 measurements and placed in vial filled with nitric acid and hydrochloric acid with a volume ratio of 1:3. The solution was ultrasonicated for overnight and then digested at 110 °C for 2 h. Samples were diluted

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in filtered and deionized water (10 mL) for inductively coupled plasma-atomic emission spectrometry (ICP-AES) analysis. Calibration was conducted with 0, 0.1, 1.0 and 10 ppm of Gd3+ standards. 3. Results and discussion 3.1. Synthesis and self-assembly of PAs PAs were designed as shown in Figure 2 and synthesized through Fmoc (9-fluorenylmethoxycarbonyl) chemistry solid-phase peptide synthesis based on N-Phe (Figure S3), which contains a hydrophobic peptide block with a propensity of β-sheet formation27,32 afforded by alternating phenylalanine (Phe) and lysine (Lys) residues linked to the hydrophilic N-(Fmoc-8-amino-3,6-dioxaoctyl)succinamic acid (Fmoc-PEG2-Suc-OH).33 The molecular weight of each PA was verified by MALDI-TOF/TOF mass spectrometry by comparing with a theoretical value (Figure S2). To establish the specific cell targeting function of PAs, octreotide was attached at the hydrophilic N-terminus of N-Phe, and the resultant PA named C-Phe (Figure 2). To explore the ability of PA to form a robust nanoarchitecture during aqueous self-assembly, C-Cha was prepared by the replacement of Phe in C-Phe with cyclohexylalanine (Cha), which has a larger π-value (π of Phe = 1.79, π of Cha = 2.72);34 this value indicates the hydrophobicity of amino acid side chains.35 The cyclization effect of the tumor cell-targeting segment of PA was investigated by comparing PAs with linear and cyclic octreotide blocks (drawn in blue in Figure 2). Cyclization of octreotide can enhance the biological stability against enzymatic hydrolysis because of a decreased structural flexibility imposed by the cyclic or β-turn conformation,36 required for the interaction with SSTR overexpressed on tumor cells.37 Next, PA with both octreotide and DOTA within a single molecule, C-Cha-DOTA, was designed, to have both target selectivity and MR efficacy. However, C-Cha-DOTA was synthesized with a low yield (8% by HPLC, Figure 2 and Figures S1 and S2), which provoked the idea of a co-assembly of different PAs, each with a different function. To

fabricate a “bottom-up” customized nanoscaffold for theranostics, C-Cha was chosen to co-assemble with DOTA-conjugated PA (Cha-DOTA). Cha-DOTA, based on the β-sheet–forming hydrophobic block of C-Cha, was synthesized using the procedure described above (Figure 2 and Figure S3). The ACS Paragon Plus Environment

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structural correspondence between the hydrophobic blocks of C-Cha and Cha-DOTA in the coassembly allows the formation of stable supramolecular nanostructures in aqueous solution.

Figure 2. Molecular structures of PAs used in this study. Reddish part: hydrophobic block, bluish part: cyclic and linear octreotide, and green part: DOTA for Gd3+-chelates.

Figure 3. a) CMCs of C-Phe and C-Cha in aqueous solution. The emission intensities of Nile red were measured at λem = 640 nm and 630 nm for C-Phe and C-Cha, respectively (λex = 550 nm). b) Fluorescence spectra indicating the Nile red-encapsulation capacity of aqueous C-Phe and C-Cha nanostructures. c) CD spectra of PAs in 0.1 м NaCl solution (0.1 mм in D2O). ACS Paragon Plus Environment

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Aqueous assembly of PAs was investigated by fluorescence spectroscopy and dynamic light scattering (DLS) measurements (Figure 3a,b and Figures S6-8) in 0.1 м NaCl solution strengthening the hydrophobic interactions between side chains in the hydrophobic peptide block by charge screening of protonated Lys residues.11 CMCs of C-Phe and C-Cha were measured by fluorescence spectroscopy using Nile red (Figure 3a and Figure S6). When self-assembled nanostructures form, the characteristic emission of Nile red can be appeared due to the location of Nile red in the hydrophobic core. As expected, C-Cha had lower CMC value (13 µм) compared with C-Phe (22 µм), which is attributed to stronger hydrophobic interactions between neighboring PAs of C-Cha. The emission of Nile redencapsulating C-Cha aggregates was blue-shifted compared with that of C-Phe (Figure 3b), which indicated that Nile red within C-Cha aggregates was located in a relatively more nonpolar environment.38 This result was also consistent with the outcomes of spectroscopic analysis of A-Phe and A-Cha with linear octreotide (Figure S7). In addition, hydrodynamic diameters of PAs, calculated by DLS using CONTIN analysis,10 had a broad distribution, suggesting formation of aggregates with a high-aspect ratio (Figure S8b). CD measurements and attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy assays were performed for detailed study of the secondary structure of PA self-aggregates (Figure 3c and Figure S9). CD spectra of PAs with Cha (C-Cha and A-Cha) showed a negative minimum ellipticity at ~212 nm and a positive maximum ellipticity at ~197 nm, characteristic for a typical β-sheet structure (Figure 3c). ATR-FTIR spectra of C-Cha and A-Cha further supported the presence of antiparallel β-sheet structures, as demonstrated by a strong amide I band at 1633 cm-1 and 1629 cm-1, respectively, with a weak band at 1703 cm-1 and 1696 cm-1, respectively (Figure S9).39 Characteristic peaks of random coil structures were observed in C-Phe and A-Phe (containing Phe in their hydrophobic domains) and were indicated by negative minimum ellipticity at ~196 nm and ~194 nm in the CD spectrum,34 respectively, and the amide I stretching band at 1637 cm-1 and 1644 cm-1 in the ATR-FTIR spectrum, respectively (Figure 3c and Figure S9).32 These results demonstrated that high hydrophobicity can lead to the formation of β-sheet conformation even in the absence of aromaticity. ACS Paragon Plus Environment

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The negative band at 201 nm and broad positive band at 220 nm in the CD spectra of C-Phe and A-Phe were a result of the π-π* transition and n-π* transition, respectively, by π-π stacking of aromatic side chains of Phe within the self-assembled structures.40 Direct visualization of aqueous PA self-aggregates was achieved by TEM (Figure 4). TEM analysis revealed that N-Phe formed micrometer-long NFs with a diameter of 4.0 ± 0.4 nm averaged over 40 NFs (Figure S10). Interestingly, cyclic octreotidecontaining C-Phe and C-Cha fibrils were helical, with a diameter of 4.5 ± 0.5 nm and 4.6 ± 0.3 nm, respectively (Figure 4a,b). The formation of helical NFs can be rationalized by steric hindrance of the bulky octreotide attached to the periphery of NF, which disrupts the parallel PA arrangement (Figure 4c).41,42 Indeed, A-Phe and A-Cha (with linear octreotides) had typical fibril structures with a diameter of 5.4 ± 0.6 nm and 5.2 ± 0.8 nm averaged over 40 NFs, respectively (Figure 4d,e).

Figure 4. TEM images showing negatively stained self-assembled PA nanostructures (0.1 mм) in 0.1 м NaCl solution; a) C-Phe and b) C-Cha. c) Schematic illustration of C-Cha showing the formation of helical NFs. Negatively stained TEM images of self-assembled d) A-Phe and e) A-Cha (0.1 mм). f) WAXS patterns of A-Cha and C-Cha. To better understand the stacking behavior of each PA type, WAXS was performed (Figure 4f). The X-ray scattering patterns of C-Cha show peaks at q = 0.52 Å-1 (12.0 Å) and 1.36 Å-1 (4.6 Å), expected to represent the mean inter-sheet distance and lateral distance between chains in a β-sheet ACS Paragon Plus Environment

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structure, respectively (Figure S11).43 While the lateral distance between β-sheet structure chains of ACha NF was retained, the inter-distance between β-sheets decreased to 9.6 Å. This difference can be ascribed to the presence of bulky, cyclic octreotide epitopes that are located on the NF exterior. The twisted molecular arrangement appears to have been induced to both, lessen the strain between the PAs and to maintain fibrillar assembly, resulting in the transformation of fibrils into helical structures (Figure 4c). Remarkably, supramolecular nanoassemblies of C-Cha (with enhanced hydrophobic βsheet structure) formed intertwined double helices with a helical pitch of ca. 13 nm (Figure 4b, inset). The presence of Na+ ions may have driven helical fibrils to form the coil-coil bundles.44 The synergetic effect of tilted PA arrangement imposed by the bulky octreotide epitope and salt-induced bundling of peripheral NF segments resulted in an effective packing of PAs with the propensity to form β-sheets (Figure 4c). The helical structure of C-Cha was further transformed into a tubular structure upon the addition of Nile red (Figure S13b). This morphological transition occurred because of the additional coiling of helical NFs to minimize the entropically unfavorable contact of the hydrophobic blocks with water,45 induced by a decrease in the relative hydrophilic volume after encapsulation of the Nile red within the hydrophobic domain. Consequently, the co-assembly behavior of C-Cha and Cha-DOTA was investigated in 0.1 м NaCl solution. It should be noted that pristine Cha-DOTA formed conventional 1D NFs (Figure S14). The mixture of C-Cha with Cha-DOTA successfully led to a construction of NFs whose fibril structure transformed from helical to conventional as the molar ratio of Cha-DOTA increased (Figure 5). And the antiparallel β-sheet structure within the resulted NFs decreased, which is confirmed by FTIR spectra (Figure S15a). These gradual changes of C-Cha-based NF characteristics imposed by addition of ChaDOTA might indicate that the successful co-assembly of PAs occurs. The direct evidence for coassembly of two PAs within the NFs was provided by two-dimensional nuclear magnetic resonance (2D NMR) spectroscopy (Figure S16).28,46 The nuclear Overhauser effect spectroscopy (NOESY) of C-Cha and Cha-DOTA mixtures unambiguously shows the intermolecular contacts between the threonine proton (Thr-γCH3) of C-Cha and the ethylenic proton of DOTA group of Cha-DOTA, indicating that ACS Paragon Plus Environment

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the NFs are made of co-assembly of PAs. The hydrophobic fluorescent dye, Nile red, was encapsulated within the co-assembled NFs of Cha-DOTA and C-Cha (Figure S15b).

Figure 5. TEM images of negatively stained co-assembled nanostructures of Cha-DOTA and C-Cha with the molar ratio of a) 2:8, b) 5:5, and c) 8:2 in aqueous solution (0.1 mм) in 0.1 м NaCl solution. 3.2. “All-in-one” workstation for targeted theranostics To evaluate the compatibility of constructed NFs in theranostic applications, cytotoxicity of NFs was investigated against HeLa cells by CCK-8 assay. HeLa cells express considerable amounts of SSTR2 that binds octreotide with high affinity.31,47,48 No noticeable toxicity was detected when various NFs decorated with octreotide (50 µм; C-Phe, C-Cha, A-Phe, and A-Cha) were incubated with HeLa cells for 24 h (Figure S17a). The pre-assessment of NFs as nanovehicles was also conducted with HeLa cells and Nile red as a water-insoluble drug model, by CLSM (Figure S18). Strong fluorescence associated with Nile red-loaded NFs was observed in the cytoplasm, confirming internalization of NFs. To study specific tumor-targeted intracellular drug delivery with NFs in more detail, two different cell lines were employed: MCF-7 and CHO-K1.47 MCF-7 cells overexpress SSTR2 on the cell surface, in contrast with much lower amounts of SSTR2 produced by CHO-K1 cells.48,49 Before testing tumor cell-specific drug delivery with NF, targeted internalization of octreotide was assessed in both cell lines. To easily identify cellular uptake, octreotide was labelled with FITC (Figure S5). Strong FITC fluorescence was clearly observed in the cytoplasm of MCF-7 cells, revealing the cellular uptake of ACS Paragon Plus Environment

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octreotide via receptor-mediated endocytosis (Figure S19).50,51 This is in contrast with the result obtained with CHO-K1 cells that showed weaker intracellular fluorescence of FITC (Figure S19).

Figure 6. a) Flow cytometry histogram of MCF-7 cells incubated with DOX-loaded NFs of C-Cha and Cha-DOTA:C-Cha with 5:5 molar ratio for 2 h and (inset) measured fluorescence intensity of DOX in MCF-7 cell. CLSM images of b-d) MCF-7 and e-g) CHO-K1 cells treated with DOX-loaded NFs (25 µм) co-assembled from Cha-DOTA and C-Cha with 5:5 molar ratio (24 h after treatment, scale bar = 50 µm): left, DOX colored red; middle, nuclei stained blue with DAPI; right, merged images. Based on these results, receptor-specific intracellular drug delivery of NFs was also assessed in the two cell lines using DOX. Several factors ascribed to the molecular structures of PA were considered for this experiment: 1) the presence of octreotide on surface of NF and its cyclization effect on cellular uptake of NF, and 2) the hydrophobicity of fibril core and drug loading capacity of NF. The characteristic fluorescence of DOX loaded in NF was utilized for tracking of NFs (Figure S20). The drug loading contents (DLCs) of N-Phe, C-Phe, C-Cha, and Cha-DOTA:C-Cha = 5:5 determined by UV-Vis spectroscopy were 5.3%, 6.7%, 8.0% and 7.5%, respectively (Figure S21a). As expected, CCha NFs exhibited a higher DLC value than C-Phe ones. The calculated drug loading efficiency (DLE) of C-Cha showed the highest value (76.6%) among the NFs (Figure S21a) which is attributed to the strong hydrophobic interactions between DOX and C-Cha within the NF core. The in vitro DOXrelease experiments were carried out under pH 5.5 sodium acetate buffer solution containing 10% DMSO at 37 °C (Figure S21b). Under acidic condition, the destructive hydrophobic interactions by ACS Paragon Plus Environment

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reprotonation of amino group of DOX resulted in the increased water solubility of DOX.52 A free DOX solution was treated as a control and was fully released in 10 h, while DOX within NFs exhibited a more sustained release. The cumulative DOX-release amounts from C-Cha NFs and C-Phe NFs reached up to 91% and 86% over a 70 h-period, respectively. Cellular uptake of DOX-loaded NFs was verified by CLSM using N-Phe, C-Phe, C-Cha, A-Phe, and A-Cha (Figures S22-24). MCF-7 cells incubated with DOX-loaded NFs of C-Cha showed the strongest fluorescence of DOX (λex = 488 nm) compared with others, indicating the highest cellular uptake of those NFs (Figure S22c). Intracellular DOX delivery of C-Cha was quantitatively evaluated by fluorescence-activated cell sorting analysis (FACS). The measured fluorescence intensity of DOXloaded C-Cha NF was ~4.5-fold higher in MCF-7 cells than the control (Figure 6a). Fluorescence of DOX-loaded NFs of A-Cha was lower in comparison with C-Cha due to insufficient interactions between the linear epitope with SSTR2 on MCF-7 cell surface (Figure S22e). In contrast to the result obtained with MCF-7 cells, no significant emission of DOX was observed in CHO-K1 cells (Figure S23). Notably, NFs of N-Phe, without octreotide, resulted in no discernable differences in DOX fluorescence between the two cell lines (Figures S22a and S23a). Therefore, we concluded that the presence of cyclic octreotide on NF surface for receptor-mediated endocytosis49,50 and the strongly hydrophobic fibrillar core for drug loading enable specific tumor-targeting of NF-based therapeutic agents. The use of NFs co-assembled from Cha-DOTA and C-Cha with 5:5 molar ratio led to fluorescence of DOX only in MCF-7 cells, as anticipated (Figures 6b-d). The fluorescence intensity of DOX was calculated to be ~8.5-fold higher than in control (Figure 6a). When MCF-7 and CHO-K1 cells were incubated with DOX-loaded NFs of Cha-DOTA and C-Cha (5:5 molar ratio, 50 µм) as a function of time, MCF-7 cell viability was 72% after 24 h, and 49% after 48 h (Figure S26a,b), indicating therapeutic effect of the co-assembled NFs delivering an anticancer drug. On the other hand, the viability of CHO-K1 cells incubated with DOX-loaded NFs was not noticeably decreased after further ACS Paragon Plus Environment

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incubation (Figure S26c,d). This indicated that DOX-loaded NFs formed by the co-assembly of ChaDOTA and C-Cha possess the tumor cell-targeting therapeutic ability. To explore the diagnostic application of co-assembled NFs, the ability of Gd3+-chelating coassembled NFs to act as a T1 MRI agent was investigated using Cha-DOTA and C-Cha mixtures with different molar PA ratios. It is well-known that Gd3+-based T1 agents are used to reduce the spin-lattice relaxation times of adjacent water molecule, increasing the signal from protons and enabling the resultant voxel seem “brighter” in T1 weighted images.25,26 Proton relaxivity (r1) of a T1 agent can be defined by the increase of longitudinal water proton relaxation rate (R1 = 1/T1, s-1) per Gd3+ concentration (mм).25,53 Before evaluating the r1 of co-assembled NFs with Cha-DOTA and C-Cha, the morphology of NFs was verified after Gd3+-chelation. No morphological change from the original NFs was observed (Figure 7a). The amount of Gd3+ chelated by the co-assembled NFs was determined by ICP-AES.37 The measured r1 values for NFs co-assembled from C-Cha and Cha-DOTA at 2:8, 5:5, and 8:2 molar ratios were determined from the slopes of linear fits as 17.2 mм-1·s-1, 17.1 mм-1·s-1, and 19.5 mм-1·s-1, respectively (Figure 7b). These calculated r1 values were significantly higher than that reported for single molecular Gd3+-DOTA contrast agents (~4 mм-1·s-1).25 A supramolecular nanoagent based on the molecular self-assembly of PAs was able to enhance the water proton relaxation rates.54 Interestingly, r1 value of co-assembled Cha-DOTA:C-Cha NFs with 2:8 molar ratio that chelated Gd3+ was slightly higher than those of NFs co-assembled with 5:5 and 8:2 PA molar ratios. This may be explained by the strong tendency of C-Cha to form a helical arrangement. In general, the amount of Gd3+ chelated by a molecular contrast agent is important in determining r1 values. However, for supramolecular contrast agents, not only the aggregation size of Gd3+-chelating self-assembled nanostructures54,55 but also steric constraints of Gd3+-chelating pendants immobilized on nanostructures56 should be considered. The average hydrodynamic diameters of co-assembled NFs from Cha-DOTA and C-Cha with Gd3+, as determined by DLS, showed a trend to decrease with increasing amounts of Cha-DOTA (Figure S27), and the tendency of PAs to form helical arrangements also ACS Paragon Plus Environment

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accordingly decreased (Figure 5). Higher molar masses of Gd3+-chelating NF agents and larger sizes slow the rotational correlation time.26 The rotational motion of Gd3+-chelates is further restricted by DOTA in the helical fibrillar structure, resulting in increased r1 values and MRI contrast.54-56 Therefore, we anticipate that the performance of supramolecular nanostructure contrast agents could be improved by rational design of molecules and precise control of their self-assembly.57

Figure 7. a) TEM image of negatively stained, co-assembled nanostructures of Cha-DOTA and C-Cha with 2:8 molar ratio after Gd3+-conjugation (0.1 mм, [Gd3+] = 1.0 mм). b) T1 relaxivity plots of Gd3+chelating NFs of Cha-DOTA:C-Cha with different molar ratios, from 2:8, 5:5, to 8:2, as a function of [Gd3+] (4.7 T, 25 °C), and (inset) T1 weighted MR images of 5:5 Cha-DOTA:C-Cha. c) T1 relaxation time measurements in mixed Cha-DOTA and C-Cha solutions as a function of the added Gd3+. MRI of Gd3+-labelled NFs co-assembled from Cha-DOTA and C-Cha (5:5 molar ratio) was performed with Gd3+ concentrations ranging from 0 to 1.0 mм (Figure 7b, inset). The most intense signal was observed at highest Gd3+ concentration (1.0 mм). To determine the optimum Gd3+ loading, T1 relaxation times of co-assembled NFs with different molar ratios of Cha-DOTA:C-Cha were measured as a function of added Gd3+ (Figure 7c). The total concentration of Cha-DOTA and C-Cha was kept constant at 0.1 mм. After the addition of ten Gd3+ equivalents, T1 relaxation time of NFs co-assembled from 20 mol% Cha-DOTA was shorter than those of NFs with 50 mol% and 80 mol% Cha-DOTA. Furthermore, T1 relaxation time became shorter with an increasing Gd3+ concentration. However, the addition of only ten Gd3+ equivalents resulted in a sufficiently short T1 relaxation time (170~195 ms) to generate significant contrast in MRI. Notably, post-addition of Gd3+ into a co-assembled NF solution ACS Paragon Plus Environment

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requires relatively large amounts of Gd3+ for chelation because of the inaccessibility of the Gd3+ to DOTA ligand attached on the surface of NFs decorated together with octreotide. 4. Conclusions A facile approach for the preparation of customized functional nanomaterials has been here developed through co-assembly of biologically active amphiphiles. In particular, supramolecular theranostic nanoagent was fabricated using a PA-fibrillar nanoplatform. Molecular scaffolds for theranostics were designed by incorporating of tumor cell-targeting and imaging moieties into co-assembled PAs. PAs based on a β-sheet–forming hydrophobic block self-assembled into fibrillar nanostructures in aqueous solution, leading to the formation of a robust nanoplatform, for co-assembly with PAs that possess different biomedical functions. The resulting co-assembled NFs were successfully used in targeted intracellular drug delivery, with high T1 relaxivity (> 10 mм-1·s-1) for MRI contrast. Notably, theranostic ability was shown to be deeply correlated with the molecular packing arrangement of the NFs. In conclusion, the supramolecular PA nanostructure is a promising candidate for a new class of theranostic agent. This work provides a useful strategy for “bottom-up” on demand customization of nanomaterial fabrication from constructed libraries containing molecular scaffolds with a variety of functions. ASSOCIATED CONTENT Supporting Information Synthesis and identification of a series of PAs, self-assembly and coassembly behavior of PAs into NFs, and their target-selective intracellular delivery. This materials is available free of charge via Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes ACS Paragon Plus Environment

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The authors declare no competing financial interest. Acknowledgments This research was supported by Basic Science Research Program (2016R1A2B4012322) and Global PH.D Fellowship Program (2015H1A2A1034643) through the National Research Foundation of Korea (NRF), and the Korea Basic Science Institute grant (D36402). References (1) Webber, M. J.; Kessler, J. A.; Stupp, S. I. J. Intern. Med. 2010, 267, 71-88. (2) Matson, J. B.; Stupp, S. I. Chem. Commun. 2012, 48, 26-33. (3) Lutolf, M. P.; Hubbell, J. A. Nat. Biotechnol. 2005, 23, 47-55. (4) Langer, R.; Tirrell, D. A. Nature 2004, 428, 487-492. (5) Dehsorkhi, A.; Gouveia, R. M.; Smith, A. M.; Hamley, I. W.; Castelletto, V.; Connon, C. J.; Reza, M.; Ruokolainen, J. Soft Matter 2015, 11, 3115-3124. (6) Branco, M. C.; Schneider, J. P. Acta Biomaterialia 2009, 5, 817-831. (7) Moyer, T. J.; Kassam, H. A.; Bahnson, E. S. M.; Morgan, C. E.; Tantakitti, F.; Chew, T. L.; Kibbe, M. R.; Stupp, S. I. Small 2015, 11, 2750-2755. (8) Lock, L. L.; Reyes, C.; Zhang, P. Cui, H. J. Am. Chem. Soc. 2016, 138, 3533-3540. (9) Geng, Y.; Dalhaimer, P.; Cai, S.; Tsai, R.; Tewari, M.; Minko, T.; Discher, D. E. Nat. Nanotechnol. 2007, 2, 249-255. (10) Park, M.-K.; Jun, S.; Kim, I.; Jin, S.-M.; Kim, J.-G.; Shin, T. J.; Lee, E. Adv. Funct. Mater. 2015, 25, 45704579. (11) Cui, H.; Webber, M. J.; Stupp, S. I. Biopolymers 2010, 94, 1-18. (12) Xie, J.; Lee, S.; Chen, X. Adv. Drug Delivery Rev. 2010, 62, 1064-1079. (13) Ling, D.; Park, W.; Park, S.-j.; Lu, Y.; Kim, K. S.; Hackett, M. J.; Kim, B. H.; Yim, H.; Jeon, Y. S.; Na, K.; Hyeon, T. J. Am. Chem. Soc. 2014, 136, 5647-5655. (14) Cho, K.; Wang, X.; Nie, S.; Chen, Z. G.; Shin, D. M. Clin. Cancer Res. 2008, 14, 1310-1316. (15) Shubayev, V. I.; Pisanic 2nd, T. R.; Jin, S. Adv. Drug Delivery Rev. 2009, 61, 467-477.

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(16) Chen, F.; Hong, H.; Goel, S.; Graves, S. A.; Orbay, H.; Ehlerding, E. B.; Shi, S.; Theuer, C. P.; Nickles, R. J.; Cai, W. ACS Nano 2015, 9, 3926-3934. (17) Du, W.; Yuan, Y.; Wang, L.; Cui, Y.; Wang, H.; Xu, H.; Liang, G. Bioconjugate Chem. 2015, 26, 2571-2578. (18) Liu, J.-N.; Bu, W.-B.; Shi, J.-L. Acc. Chem. Res. 2015, 48, 1797-1805. (19) Chen, W.; Xu, N.; Xu, L.; Wang, L.; Li, Z.; Ma, W.; Zhu, Y.; Xu, C.; Kotov, N. A. Macromol. Rapid Commun. 2010, 31, 228-236. (20) Harris, A. G. Gut 1994, 35, S1-S4. (21) Accardo, A.; Aloj, L.; Aurilio, M.; Morelli, G.; Tesauro, D. Int. J. Nanomedicine 2014, 9, 1537-1557. (22) Zhou, S.; Wu, Z.; Chen, X.; Jia, L.; Zhu, W. ACS Appl. Mater. Interfaces 2014, 6, 11459-11469. (23) Lauffer, R. B. Chem. Rev. 1987, 87, 901-927. (24) Bull, S. R.; Guler, M. O.; Bras, R. E.; Venkatasubramanian, P. N.; Stupp, S. I.; Meade, T. J. Bioconjugate Chem. 2005, 16, 1343-1348. (25) Preslar, A. T.; Parigi, G.; McClendon, M. T.; Sefick, S. S.; Moyer, T. J.; Haney, C. R.; Waters, E. A.; MacRenaris, K. W.; Luchinat, C.; Stupp, S. I.; Meade, T. J. ACS Nano 2014, 8, 7325-7332. (26) Diaferia, C.; Gianolio, E.; Palladino, P.; Arena, F.; Boffa, C.; Morelli, G.; Accardo, A. Adv. Funct. Mater. 2015, 25, 7003-7016. (27) Zhao, X.; Zhang, S. Chem. Soc. Rev. 2006, 35, 1105-1110. (28) Niece, K. L.; Hartgerink, J. D.; Donners, J. J. J. M.; Stupp, S. I. J. Am. Chem. Soc. 2003, 125, 7146-7147. (29) Song, A.; Wang, X.; Zhang, J.; Mařík, J.; Lebrilla, C. B.; Lam, K. S. Bioorg. Med. Chem. Lett. 2004, 14, 161-165. (30) Morisco, A.; Accardo, A.; Gianolio, E.; Tesauro, D.; Benedetti, E.; Morelli, G. J. Pept. Sci. 2009, 15, 242250. (31) Abdellatif, A. A. H. Biochem. Physiol. 2015, 4, 1000183. (32) Bowerman, C. J.; Liyanage, W.; Federation, A. J.; Nilsson, B. L. Biomacromolecules 2011, 12, 2735-2745. (33) Lamm, M. S.; Rajagopal, K.; Schneider, J. P.; Pochan, D. J. J. Am. Chem. Soc. 2005, 127, 16692-16700. (34) Bowerman, C. J.; Ryan, D. M.; Nissan, D. A.; Nilsson, B. L. Mol. BioSyst. 2009, 5, 1058-1069. (35) Fauchère, J.-L.; Charton, M.; Kier, L. B.; Verloop, A.; Pliska, V. Int. J. Pept. Protein Res., 1988, 32, 269278. ACS Paragon Plus Environment

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(36) Tugyi, R.; Mezö, G. Fellinger, E.; Andreu, D. Hudecz, F. J. Peptide Sci. 2005, 11, 642-649. (37) Accardo, A.; Morisco, A.; Gianolio, E.; Tesauro, D.; Mangiapia, G.; Radulescu, A.; Brandt, A.; Morelli, G. J. Pept. Sci. 2011, 17, 154-162. (38) Greenspan, P.; Fowler, S. D. J. Lipid Res. 1985, 26, 781-789. (39) Wibowo, S. H.; Sulistio, A.; Wong, E. H. H.; Blencowe, A.; Qiao, G. G. Adv. Funct. Mater. 2015, 25, 31473156. (40) Yang, Z.; Liang, G.; Ma, M.; Abbah, A. S.; Lu, W. W.; Xu, B. Chem. Commun. 2007, 843-845. (41) Tsai, W.-W.; Li, L.-s.; Cui, H.; Jiang, H.; Stupp, S. I. Tetrahedron 2008, 64, 8504-8514. (42) Ghosh, A.; Haverick, M.; Stump, K.; Yang, X.; Tweedle, M. F.; Goldberger, J. E. J. Am. Chem. Soc. 2012, 134, 3647-3650. (43) Maity, I.; Mukherjee, T. K.; Das, A. K. New J. Chem. 2014, 38, 376-385. (44) Lee, E.; Hammer, B.; Kim, J.-K.; Page, Z.; Emrick, T.; Hayward, R. C. J. Am. Chem. Soc. 2011, 133, 1039010393. (45) Mai, Y.; Eisenberg, A. Chem. Soc. Rev. 2012, 41, 5969-5985. (46) Behanna, H. A.; Donners, J. J. J. M.; Gordon, A. C.; Stupp, S. I. J. Am. Chem. Soc. 2005, 127, 1193-1200. (47) Buttke, T. M.; McCubrey, J. A.; Owen, T. C. J. Immunol. Methods 1993, 157, 233-240. (48) Tian, X.; Baek, K.-H.; Shin, I. Chem. Sci. 2013, 4, 947-956. (49) Sun, M.; Wang, Y.; Shen, J.; Xiao, Y.; Su, Z.; Ping, Q. Nanotechnology 2010, 21, 475101-475111. (50) Lelle, M.; Kaloyanova, S.; Freidel, C.; Theodoropoulou, M.; Musheev, M.; Niehrs, C.; Stalla, G.; Peneva, K. Mol. Pharmaceutics 2015, 12, 4290-4300. (51) Huang, C.-M.; Wu, Y.-T.; Chen, S.-T. Chem. Biol. 2000, 7, 453-461. (52) Shi, F.; Ding, J.; Xiao, C.; Zhuang, X.; He, C.; Chen, L.; Chen, X. J. Mater. Chem. 2012, 22, 14168-14179. (53) Laus, S.; Sour, A.; Ruloff, R.; Tóth, E.; Merbach, A. E. Chem.-Eur. J. 2005, 11, 3064-3076. (54) Liu, S.; Zhang, P.; Banerjee, S. R.; Xu, J.; Pomper, M. G.; Cui, H. Nanoscale 2015, 7, 9462-9466. (55) Manus, L. M.; Mastarone, D. J.; Waters, E. A.; Zhang, X.-Q.; Schultz-Sikma, E. A.; MacRenaris, K. W.; Ho, D.; Meade, T. J. Nano Lett. 2010, 10, 484-489. (56) Goswami, L. N.; Ma, L.; Chakravarty, S.; Cai, Q.; Jalisatgi, S. S.; Hawthorne, M. F. Inorg. Chem. 2013, 52, 1694-1700. ACS Paragon Plus Environment

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(57) Preslar, A. T.; Tantakitti, F.; Park, K.; Zhang, S.; Stupp, S. I.; Meade, T. J. ACS Nano 2016, 10, 7376-7384.

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Table of contents 74x51mm (300 x 300 DPI)

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Figure 1. Schematic illustration of an “all-in-one” 1D supramolecular nanoplatform prepared by co-assembly of functional PAs for theranostic purposes. 152x110mm (300 x 300 DPI)

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Figure 2. Molecular structures of PAs used in this study. Reddish part: hydrophobic block, bluish part: cyclic and linear octreotide, and green part: DOTA for Gd3+-chelates. 119x119mm (300 x 300 DPI)

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Figure 3. a) CMCs of C-Phe and C-Cha in aqueous solution. The emission intensities of Nile red were measured at λem = 640 nm and 630 nm for C-Phe and C-Cha, respectively (λex = 550 nm). b) Fluorescence spectra indicating the Nile red-encapsulation capacity of aqueous C-Phe and C-Cha nanostructures. c) CD spectra of PAs in 0.1 м NaCl solution (0.1 mм in D2O). 207x53mm (300 x 300 DPI)

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Figure 4. TEM images showing negatively stained self-assembled PA nanostructures (0.1 mм) in 0.1 м NaCl solution; a) C-Phe and b) C-Cha. c) Schematic illustration of C-Cha showing the formation of helical NFs. TEM images of self-assembled d) A-Phe and e) A-Cha (0.1 mм). f) WAXS patterns of A-Cha and C-Cha. 186x92mm (300 x 300 DPI)

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Figure 5. TEM images of negatively stained co-assembled nanostructures of Cha-DOTA and C-Cha with the molar ratio of a) 2:8, b) 5:5, and c) 8:2 in aqueous solution (0.1 mм) in 0.1 м NaCl solution. 181x52mm (300 x 300 DPI)

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Figure 6. a) Flow cytometry histogram of MCF-7 cells incubated with DOX-loaded NFs of C-Cha and ChaDOTA:C-Cha with 5:5 molar ratio for 2 h and (inset) measured fluorescence intensity of DOX in MCF-7 cell. CLSM images of b-d) MCF-7 and e-g) CHO-K1 cells treat 57x20mm (300 x 300 DPI)

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Figure 7. a) TEM image of negatively stained, co-assembled nanostructures of Cha-DOTA and C-Cha with 2:8 molar ratio after Gd3+-conjugation (0.1 mм, [Gd3+] = 1.0 mм). b) T1 relaxivity plots of Gd3+chelating NFs of Cha-DOTA:C-Cha with different molar ratios, from 2:8, 5:5, to 8:2, as a function of [Gd3+] (4.7 T, 25 °C), and (inset) T1 weighted MR images of 5:5 Cha-DOTA:C-Cha. c) T1 relaxation time measurements in mixed Cha-DOTA and C-Cha solutions as a function of the added Gd3+. 204x51mm (300 x 300 DPI)

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