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Supramolecular self-assemblies with nanoscale RGD clusters promote cell growth and intracellular drug delivery Fengyang Xu, Jie Liu, Jian Tian, Lingfeng Gao, Xiaju Cheng, Yue Pan, Ziling Sun, and Xinming Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b08624 • Publication Date (Web): 19 Oct 2016 Downloaded from http://pubs.acs.org on October 19, 2016

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ACS Applied Materials & Interfaces

Supramolecular Self-Assemblies with Nanoscale RGD

Clusters

Promote

Cell

Growth

and

Intracellular Drug Delivery Fengyang Xu,† Jie Liu,† Jian Tian, ‡ Linfeng Gao,† Xiaju Cheng,‡ Yue Pan,† Ziling Sun*,‡ and Xinming Li*,†, †

⊥

State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials,

College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, China ‡

School of Biology and Basic Medical Science, Soochow University, Suzhou, 215123, China

⊥

State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai

200433, China KEYWORDS: hydrogel; peptide; self-assembly; supramolecular; multivalence.

ABSTRACT: In this work, we reported the generation of a novel supramolecular hydrogelator from a peptide derivative which consisted of a structural motif (e.g., Fc-FF) for supramolecular self-assembly and a functional moiety (e.g., RGD) for integrin binding. Following self-assembly in water at neutral pH, this molecule firstly tended to form metastable spherical aggregates,

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which subsequently underwent a morphological transformation to form high-aspect-ratio nanostructures over 2 h when aged at room temperature. More importantly, because of the presence of nanoscale RGD clusters on the surface of nanostructures, the self-assembled nanomaterials (e.g., nanoparticles and nanofibers) can be potentially used as a biomimetic matrix for cell culture, and as a vector for cell-targeting drug delivery via multivalent RGD-integrin interactions.

1. INTRODUCTION Cell-material interactions played a crucial role in the field of tissue engineering and biomedicine, because they can not only direct tissue formation by modulating cellular functions in vitro and in vivo such as cell adhesion, locomotion, growth, proliferation and differentiation,1-2 but also affect the efficacy of intracellular trafficking of delivered drugs by carriers.3 Therefore, modification of material surfaces with different cell-adhesive ligands represents a major discipline in the preparation of cell-association biomaterials.4 Among different molecules for cell association, the amino acid sequence Arg-Gly-Asp (RGD) is one of the highly effective cell recognition motifs for material surface modification to achieve integrin-mediated cell interaction.5 Integrin, as heterodimeric transmembrane cell surface receptors expressed by most cell types, consists of an α and a β subunit to mediate cell adhesion to extracellular matrixes (ECM) by interacting with RGD ligands in fibronectin, vitronectin, fibrinogen and collagen.6 In addition, integrin was also identified to be overexpressed by numerous solid tumors and served as a marker of angiogenesis, tumor development and metastasis.7 Therefore, integrin can be used as an important target for designing biomimetic materials with improved cell adhesion and migration properties,8-12 or preparing drug delivery systems for cancer diagnosis and therapy.13-15


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Although the binding affinity between monomeric RGD ligand and integrin receptor is relatively weak, the organization of RGD ligands on the surface of materials to form nanoscale clusters can enhance cell adhesion and direct cell responses.16-20 Self-assembly is a spontaneous process to organize molecules into the formation of larger aggregates.21-22 Owing to numerous advantages, such as easily synthesized small molecules, well-defined molecular structures of building blocks and spontaneous process of supramolecular assembling, self-assembly has become an efficient strategy for the preparation of nanomaterials with nanoscale precision.23-27 Therefore, integrating a structural motif possessing self-assembling properties with a cell adhesive ligand (e.g., RGD) would offer an attractive method for preparing novel biomaterials with spatially orienting RGD ligands on their surfaces for integrin binding and cell-material recognition. In addition, biomaterials prepared from self-assembled peptides also exhibited several unique advantages, such as high biocompatibility, inherent bioactivity and biodegradability, and dynamic and adaptable nature of the formed materials. 28-32 Herein, we designed and synthesized a new peptide analogue (Fc-FFRGD) which consisted of a ferrocene group (Fc), a Phe-Phe dipeptide segment and a RGD moiety, and explored its potentials for the formation of novel biomaterials for cell-material interaction (Scheme 1). The Fc group and Phe-Phe dipeptide segment increased the rigidity of scaffold and provided driving forces for supramolecular self-assembly via hydrophobic and aromatic-aromatic interactions.33-35 Although ferrocene carboxylic acid has been previously employed for the design and synthesis of new supramolecular gelator with self-assembling and morphological transition properties,36 its potentials to work as a new biomimetic material for cell adhesion and growth or a nanocarrier for drug encapsulation and delivery were less explored. The RGD moiety offered the self-assembled nanomaterials with the capability to interact with cell-surface integrins via multivalent RGD-


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integrin bindings. Owing to the typical amphiphilicity of molecular structure together with strong non-covalent interactions, this molecule can perform supramolecular self-assembly in water and form stable nanofibrillar structures and a hydrogel with a minimum gelation concentration of 1.0 wt%. Due to the presence of multiple RGD ligands on the surface of nanofibrillar structures, this supramolecular hydrogel exhibited high potentials to work as a new biomimetic scaffold to promote integrin-dependent cell adhesion and proliferation. On the other hand, at a much lower concentration (0.1 wt%), Fc-FFRGD tended to form stable nanospherical structures. And the self-assembled nanoparticles from Fc-FFRGD were also suitable for hydrophobic drug encapsulation and targeting drug delivery, because of the formation of nanospherical structure with a hydrophobic inner core, nanoscale RGD clusters on its outer surface and stimuliresponsive property towards the pH change of solution. Therefore, the rational organization of a rigid and hydrophobic group (e.g., Fc-FF) with a hydrophilic peptide segment (e.g., RGD) can offer a simple approach to prepare biofunctional supramolecular gelator and biomaterials with cell-interaction ability. 2. MATERIALS AND METHODS 2-Chlorotrityl chloride resin, Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH and FmocAsp(OtBu)-OH were purchased from Shanghai GL Biochem. Ferrocenecarboxylic acid, 0.1% triton X-100, fluorescein-phalloidin, DAPI and staining dyes for live/dead cell assays were provided by Sigma-aldrich. All the other raw materials were obtained from Sigma and J&K Chemical without further purification unless mentioned. Fc-FFRGD was synthesized through solid-phase peptide synthesis (SPPS). 1H NMR spectra were obtained on a Unity Inova 400 by using DMSO-d6 as a solvent. CD spectroscopy study was performed on a JASCO J-810 spectrometer. LC-MS analysis was conducted on an Agilent 6120 Quadrupole LC/MS system


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with an ESI resource. HPLC purification and analysis were carried out on a Waters 600E Multisolvent Delivery System using a YMC C18 RP column with CH3OH (0.1% of TFA) and water (0.1% of TFA) as eluents. Transmission electron micrograph (TEM) and scanning electron micrograph (SEM) images were recorded on a Tecnai G220 transmission electron microscope and a Hitachi S-4800, respectively. Fluorescence microscopy images were taken on a IX71 Olympus microscope, Japan. Fourier transform infrared spectroscopy (FTIR) characterization was carried out on a Perkin-Elmer spectrophotometer. Rheological study was taken on a Thermo Scientific HAAKE RheoStress 6000 rheometer. Synthesis of Fc-FFRGD hydrogelator Fc-FFRGD was synthesized by following SPPS protocols with the application of FmocAsp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH and ferrocenecarboxylic acid. Firstly, 2-chlorotrityl chloride resin (1.0 g, 1.3-1.8 mmol/g) was dissolved in dry DCM with N2 bubbling for 0.5 h, and the swelled resin was washed by dry DMF. Afterwards, the mixed solution containing the first amino acid (Fmoc-Asp(OtBu)-OH) and N,N-diisopropylethylamine (DIPEA) was added to reactor containing swelled resin, and reacted for 1 h. After removal of solvent, the resin was washed by dry DMF and quenched by blocking solution (80:15:5 of DCM/methanol/DIEA). Then, the resin was treated with 20% piperidine (in DMF) for 0.5 h, and washed thoroughly with DMF. The subsequent Fmoc-protected amino acid (e.g., Fmoc-Gly-OH) was added to the reactor and coupled with free amino groups on the resin by using HBTU as a coupling reagent. These coupling and deprotection steps were repeated by following standard Fmoc SPPS protocol to elongate peptide chain. Then the synthetic peptide was cleaved from the resin by using TFA cocktail solution (90% TFA in water), and the resulted product was purified by HPLC by using water-methanol as eluents (from 80:20 to 0:100) to afford Fc-FFRGD in


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yield of 72%. 1H NMR (400 MHz, DMSO-d6): δ8.18-8.08 (m, 5H), 7.87 (d, 3H), 7.42-7.19 (m, 10H), 4.85-4.73 (m, 5H), 3.91 (s, 5H), 4.34 (s, 4H), 3.06 (d, 4H), 2.96-2.90 (m, 4H), 1.54-1.52 (m, 4H). MS: calcd M = 853.3, obsd (M-H)- = 852.3. Circular dichroism and FTIR characterization The Fc-FFRGD hydrogel (0.2 mL, 1.0 wt%) was placed evenly on a quartz cuvette of 1 mm thickness and scanned from 185 to 400 nm by using a JASCO J-810 spectrometer under N2 atmosphere. FTIR spectra were collected on a Perkin-Elmer spectrophotometer by loading FcFFRGD hydrogel (1.0 wt%, pH=7.4) into a KBr cuvette. The hydrogel was prepared by using deuterium oxide (D2O) as a solvent, and deuterium chloride (DCl) and deuterium generation of NaOH (NaOD) as an acid and a base, respectively. Rheological tests 300 µL of Fc-FFRGD hydrogel was loaded on a parallel plate (20 mm). The dynamic strain sweep test was run from 0.1 to 10% strain with frequency at 6.282 rad/s at 25 oC on a Thermo Scientific HAAKE ReoStress 6000 rheometer. The dynamic frequency sweep test was run from 100 to 0.1 rad/s, and a strain at 0.5% was used to ensure the linearity of dynamic viscoelasticity. Dynamic light scattering: The hydrodynamic radius and size distribution of the self-assembled nanostructures of Fc-FFRGD were obtained from dynamic light scattering (Brookhaven Instruments: BI-200SM goniometer and BI-9000AT autocorrelator) with a He-Ne laser (Spectra Physics, model 127 with 35 mW at 632.8 nm) and an avalanche photodiode detector (BI-APD). The scattered light was collected at an angle of 90° under controlled temperature (0.1 °C). The autocorrelation functions were analyzed by Laplace inversion (CONTIN). Live-dead assay


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Prior to cell seeding, a 96-well plate was coated with 100 µL of Fc-FFRGD hydrogel (1.0 wt%, pH=7.4), and covered with 100 µL of DMEM (Dulbecco's modified Eagle's) medium for buffer exchange. The medium in the wells was then replaced with fresh medium after 12 h incubation. Then 100 µL of cell suspension in complete medium containing 4.0 x 104 cells/mL was pipetted into each well. The 96-well plate was placed in incubator at 37 °C under 5% of CO2 atmosphere, and the medium was changed every other day. The cultured cells were stained by calcein/ethidium homodimer and imaged by OLYMPUS IX71 fluorescence microscope. Microfilament staining After 1 day in culture, the living cells on gel were washed by PBS buffer (pH=7.4), followed by the addition of 4% paraformaldehyde for cell fixation. After incubation at room temperature for 30 min, the cell-seeded gels were washed by 0.1% triton X-100 in PBS buffer twice (5 mins for each time). Finally, the cells were treated by FITC-phalloidin (40 µg/mL) and counterstained with DAPI (1 µg/mL) for 40 min. The stained samples were visualized by Confocal Laser Scanning Microscope with the excitation filters of 488 nm (green, FITC) and 405 nm (blue, DAPI). Integrin blocking In order to confirm that the adhesion of NIH/3T3 and HUVEC cells to Fc-FFRGD gel was via the interactions between α5β1 integrins and RGD residues, we treated NIH/3T3 (4.0 x 104 cells/mL) and HUVEC (4.0 x 104 cells/mL) cells in DMEM (1 mL) with 5 µL of anti-human α5β1-integrin antibody (J&L Biological) to block the α5β1 integrins on NIH/3T3 and HUVEC cells. After incubation at room temperature for 15 min, the suspensions of cells and antibody mixture were loaded on the surface of Fc-FFRGD gel. After incubation at 37 °C under 5% of


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CO2 atmosphere for 24 h, the cultured cells were subjected to microfilament staining, and imaged by OLYMPUS IX71 fluorescence microscope. Drug encapsulation 20 mg of Fc-FFRGD and 2 mg of doxorubicin (DOX) were dissolved in 20 mL of PBS buffer (pH 7.4), and incubated at room for 24 h to undergo supramolecular assembly and drug encapsulation. Then the mixed solution was transferred to a dialysis tube (Molecular weight cutoff: 1000 Da), and subjected to dialysis against 200 mL PBS buffer (pH 7.4) for 24 h. Finally, the solution inside the dialysis tube were dried on freeze dryer, re-dissolved in 3 mL DMSO, and analyzed with fluorescence spectrometer for the quantification of drug loading efficiency. The drug loading content (DLC) and drug loading efficiency (DLE) were calculated according to the following equations: DLC (%) = (weight of loaded DOX)/ (weight of DOX loaded nanoparticles) ×100 DLE (%) = (weight of loaded DOX)/ (weight of added DOX) × 100 In vitro drug release After DOX loading, the in vitro DOX release behaviors of Fc-FFRGD vesicles were measured at 37 ˚C in PBS buffer at different pHs (e.g., pH=7.4 and 6.0). The dialysis tubes containing Fc-FFRGD vesicles loaded with DOX were immersed into 200 mL PBS buffer at pH 7.4 and 6.0, respectively. Then 5 mL of solution from each sample was taken at each specific time and analyzed by fluorescence spectrometer with the excitation wavelength at 490 nm and emission wavelength at 588 nm. The cumulative amount of DOX released from vesicles was calculated by the formula: Cumulative DOX release (%) = MT/ML × 100%. MT represented the total amount of DOX released at time T. ML represented the total amount of DOX loaded in vesicles.


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Fluorescence polarization assay In order to determine the binding affinity of Fc-FFRGD nanostructures with integrin αvβ3, we chose 5(6)-carboxyfluorescein-c[RGDfK] as fluorescent probe and performed competitive displacement studies via fluorescence polarization assay. A newly synthesized peptide derivative (e.g., Fc-FFGRD) with scrambled RGD sequence was used as a negative control. Firstly we dissolved Fc-FFRGD and Fc-FFGRD in PBS buffer (pH 7.4), separately, at a concentration of 10 mM, followed by dilution with Tris buffer (50 mM Tris, pH 7.4, 1 mM CaCl2, 10 µM MnCl2, 1 mM MgCl2 and 100 mM NaCl) to concentrations ranging from 0.2 to 2 mM. Then we added Fc-FFRGD or Fc-FFGRD to the solutions which contain both 5(6)-carboxyfluoresceinc[RGDfK] probe (10 nM) and αvβ3 (280 nM). Finally, these mixture solutions were incubated at 29 oC for 5 min. Their fluorescence polarization values were read by Tecan polarion reader (Ex=485 nm, Em=510 nm) and recorded in mP. All data points were repeated 5 times and presented as mean values ± standard deviation. Cytotoxicity Assay Cytotoxicity of Fc-FFRGD, free DOX and Fc-FFRGD vesicles encapsulating DOX were evaluated in HeLa cell lines by using CCK-8 assay. Cells were seeded at the density of 4.0 x 104 cells/well in a 96-well plate, followed by the addition of Fc-FFRGD (1.5, 15, 25, 50, 75, 100 and 150 µM), free DOX and Fc-FFRGD vesicles loaded with DOX (both were under the concentration of DOX at 0.1, 1, 5 and 10 µM), and incubated under humidified atmosphere of 5% CO2 at 37 oC for 24 h. Then the cells were treated with cell counting kit-8 solution. Subsequently, the absorbance (optical density, OD) at 450 nm was measured using a Thermo Scientific Varioskan Flash spectral scanning multimode reader. Cell viability was expressed as a


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percentage of the number of control (untreated) cells. Viability in the control group was designated as 100%. All experiments were performed in triplicate. Intracellular uptake With the purpose of observing the intracellular uptake of free DOX and Fc-FFRGD vesicles encapsulating DOX, HeLa Cells were seeded onto glass coverslips at the density of 5.0 x 104 cells/well, followed by the addition of free DOX (10 µM) and Fc-FFRGD vesicles encapsulating DOX (under the concentration of DOX at 10 µM), respectively, and incubated under humidified atmosphere of 5% CO2 at 37 °C. After 24 h, the cells were fixed by paraformaldehyde (4%), counterstained by DAPI (1 µg/mL) and visualized by Confocal Laser Scanning Microscope with the excitation filters of 488 nm (red, Dox) and 405 nm (blue, DAPI). 3. RESULTS AND DISCUSSION By following the typical procedures of SPPS and employing 2-chlorotrityl chloride resin, we synthesized Fc-FFRGD from corresponding Fmoc-protected amino acids and ferrocene carboxylic acid. After final condensation with ferrocene acetic acid, the molecule was cleaved from resins and purified by HPLC, which afforded the compound with yield around 72%. Its molecular structure was confirmed by NMR and MS analysis (Figure S1). After getting Fc-FFRGD, we tested its self-assembling property by dissolving 3 mg of FcFFRGD in water. Then followed by the change of pH to 7.4, this sample transformed from a viscous solution to a stable hydrogel (1.0 wt%) over 2 h (Figure 1A and 1B), indicating the occurrence of supramolecular self-assembly in water by Fc-FFRGD. Rheological tests revealed that the storage modulus (G’) of Fc-FFRGD hydrogel was 5 times higher than its loss modulus (G’’) within the investigated oscillating frequency limit (0.1-200 rad/s), signifying the dominantly elastic properties of the self-assembled hydrogel (Figure 1C and 1D). These results


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indicated that the synthetic molecule containing a rigid and hydrophobic group (e.g., Fc-FF) and a hydrophilic peptide segment (e.g., RGD) can work as an effective gelator for supramolecular assembly and hydrogel formation. The gradual transition from a viscous solution to a stable gel observed in the gelation test would suggest that there existed a steady transformation in the nanostructure of Fc-FFRGD assemblies over time. In order to confirm our speculation, we used transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to monitor the morphological transition of nanostructures during the process of supramolecular assemblies. The freshly prepared solution at pH=7.4 was taken out at different times (e.g., 1 min, 10 min, 1 h, 2 h, 12 h, and 24 h), loaded on a carbon-coated copper grid and imaged by TEM. In the beginning, this molecule tended to form separately dispersed nanoparticles with size around 150 nm (Figure 2A), which was consistent with the amphiphilicity of its molecular structure. Then these nanoparticles started to adhere to each other and formed aggregates with cohered edge (Figure 2B and 2C) with time. The adjacent particles continually fused with each other (Figure 2D) to form wellordered 1-D nanofibrillar structures in 2 h, but irregularly scattered particles still existed, which adhered to the newly formed nanofibrillar structures (Figure 2E). However, after 12 h, the nanoparticles disappeared and transformed into well-defined nanofibers with width around 90 nm (Figure 2F), and its morphologies remained intact (Figure 2G). SEM examination (Figure 2H-2N) offered another view of the morphology transition of Fc-FFRGD from irregular nanoparticles to well-defined nanofibers via metastable pearl-necklace-like aggregates. And the resultant nanofibrillar structures can physically cross-link each other to form a fibrous network to support the formation of a stable supramolecular hydrogel. These results indicated that FcFFRGD preferred to form metastable nanoparticles at the beginning of self-assembling, and then


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transformed into thermodynamically stable fibrillary structures driven by electrostatic force and aromatic stacking interaction. To have a deep analysis of the sol-gel transition, dynamic light scattering (DLS) analysis and time-dependent rheology measurement were applied to monitor the self-assembling process of Fc-FFRGD in water. For example, the freshly prepared solution showed a relatively narrow size distribution, with diameter in the range of 100-200 nm, which was consistent with the formation of nanoparticles with hydrodynamic radius around 100 nm (Figure 3A). In comparison, as the aging time extended to 1 h and 24 h, respectively, two new peaks with increased intensities appeared around 500 nm and 1000 nm, signifying the formation of larger aggregates with a wide size distribution. In addition, dynamic time sweep experiment provided further information about the gelation kinetics of Fc-FFRGD solution from the measurement of its storage modulus (G’) and loss modulus (G’’) as a function of time (Figure 3B). For instance, at the beginning of gelation, the storage modulus (G’) of solution was much lower than its loss modulus (G’’), indicating the typically fluid state of the sample. As the gelation process proceeded, the storage modulus (G’) increased more rapidly than its loss modulus (G’’) due to the occurrence of supramolecular self-assembly and the formation of self-assembled nanostructures (e.g., nanoparticles and nanofibers), and a crossover point between G’ and G’’ curves was observed at 600 sec. Then the storage modulus (G’) increased constantly with time and stabilized around 10000 Pa after 1800 sec, which was much higher than the loss modulus (G’’), indicating the formation of a stable hydrogel. Circular dichroism (CD) and Fourier transform infrared spectroscopy (FTIR) studies provided insightful information about the secondary structures formed by Fc-FFRGD


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during its supramolecular self-assembly. As shown in Figure 3C, the CD spectrum of the freshly prepared Fc-FFRGD solution (e.g., 0 min) showed a negative peak at 218 nm and a positive band around 197 nm, which indicated the less-ordered state of molecules in nanoparticles.37 However, when the aged time was extended, the CD signals at these two wavelengths shifted to 203 and 220 nm, respectively, together with gradually increased intensities of CD signals at these two wavelengths. Based on the previous studies,38-39 the shifts of CD signals from 197 and 218 nm to 203 and 220 nm, respectively, shown in Figure 3C could be ascribed to re-arrangement of the molecules for the formation of twisted β-sheet structure. FTIR analysis provided additional evidence about the conformational transition of Fc-FFRGD during supramolecular self-assembly (Figure 3D). FTIR showed two major amide I peaks located at 1617 cm-1 and 1674 cm-1, respectively, at zero time (e.g., 0 min), consistent with random coil conformation, and they gradually shifted to 1630 cm-1 and 1686 cm-1 after 2 h, and stabilized at these wavelengths when the incubation time was prolonged to 24 h, suggesting the formation of an antiparallel β-sheet structure.40 These results confirmed that the observed morphological transition from nanoparticles to nanofibers in electron micrographs was closely concomitant with a secondary structural change from an unordered structure into a much more stable β-sheet structure. Since the tripeptide RGD sequence is a principal integrin-binding domain in numerous ECM proteins to promote integrin-mediated cell attachment, cell spreading, actin-skeleton formation, and focal-adhesion interaction, the introduction of RGD peptides to the inert surfaces of artificial materials can afford them the capability of integrin-dependent cell binding. Therefore, we firstly tested its potency to interact with integrin αvβ3 by using a


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fluorescence polarization assay.41-42 As revealed by Figure S2, Fc-FFRGD nanostructures exhibited obvious binding affinity with αvβ3 with EC50 around 800 µM (EC50=the concentration of ligand to reduce the fluorescence polarization value to below 50% of its initial level), in comparison with the ligand of Fc-FFGRD with scrambled RGD sequence. After confirming the binding interactions between RGD ligands of our designed nanomaterial and integrins, we further explored its potentials to interact with NIH/3T3 and HUVEC cells for cell-adhesion and proliferation, and these two types of cells have been widely used as models to examine the potency of RGD functionalized materials for cell adhesion, spreading and proliferation via integrin-RGD interactions.8, 43 Due to the good biocompatibilities of FcFFRGD gelator and the high stabilities of its hydrogel in cell culture media (Figure S3 and S4), we seeded NIH/3T3 and HUVEC cells on the surface of Fc-FFRGD hydrogels, and examined their cell adhesion and spreading properties over the course of 5-day culture. On the basis of the live-dead assays shown in Figure 4A, these two types of cells can attach and spread well on this hydrogel after 1 day incubation, as exemplified by the presence of spindle-shaped cells. Cytoskeletal F-actin staining experiments further indicated that these two types of cells formed highly-elongated and well-defined stress actin filaments (green) around their nuclei (blue) inside cells (Figure 4C and 4D), indicating the establishment of integrin-mediated cell spreading and focal adhesion interaction, whereas the cells treated with anti-human α5β1-integrin antibody lost their capability to bind RGD-ligands on the surface of Fc-FFRGD hydrogel for cell adhesion and proliferation, and the cells remained rounded morphologies with disordered F-actin inside cells (Figure S5).44-45 Due to the favorable interactions with Fc-FFRGD hydrogel via integrin-dependent bindings, these two types of cells grew rapidly, and formed cell


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clusters at day 3. With the extension of culture time, these cells tended to form confluent monolayers on the surface of hydrogels at day 5 and revealed the typical cell morphologies. Cell density quantifications further confirmed the steady increase of cell numbers of NIH/3T3 (>8.0 fold) and HUVEC (>6.8 fold) on Fc-FFRGD hydrogels over the course of cell culture (Figure 4B), while the cell densities of NIH/3T3 and HUVEC cultured on tissue culture plate increased 9.5 and 7.3 fold, respectively, at the same conditions (Figure S6 and S7). Therefore, the supramolecular hydrogel of Fc-FFRGD with ordered array of RGD ligands on the surface of self-assembled nanofibers can work as a biomimetic scaffold to aid cell adhesion, spreading and proliferation via integrinRGD interactions. Besides forming high-aspect-ratio nanostructures above minimum gelation concentration (1.0 wt%), Fc-FFRGD also exhibited the potential to form nanoparticle structure at pH 7.4 and a concentration of 0.1 wt%. In addition, the self-assembled nanoparticles exhibited stimuliresponse towards the pH change of solution, and underwent structural disassembly under acidic condition (pH=6.0). Therefore, we also checked the potentials of self-assembled nanoparticles to encapsulate drug (e.g., doxorubicin) inside their hydrophobic inner cores via hydrophobic interactions, and then performed a fast release of encapsulated drug upon exposure to the condition with acidic pH. When Fc-FFRGD was dissolved in neutral medium with a concentration at 0.1 wt%, the amphiphilic peptide can self-assemble into well-dispersed spherical nanoparticle with diameter around 30 nm. With the loading of DOX, the average diameter of nanostructure increased to 40 nm (Figure S8), due to the successful encapsulation of DOX (DLC=5.7% and DLE=32.5%), and the encapsulated drug also exhibited great potentials to stabilize the nanoparticle morphology via physical interactions (Figure S9).46-47 After confirming


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the good pH sensitivity of Fc-FFRGD nanostructure and the sustained release of encapsulated DOX (Figure S11), we further tested its capabilities for integrin binding and intracellular delivery of the encapsulated drug to cancer cells, because integrin αvβ3 were found to overexpress on the cellular membrane of certain cancer cells such as melanoma, prostate, ovarian and breast,48 which made possible selective delivery of anticancer drugs by targeting integrin αvβ3.49 Based on the fluorescence polarization assay and confocal laser scanning microscopy (CLSM) studies shown in Figure 5, we identified the existence of binding interaction between Fc-FFRGD nanoparticles and integrin αvβ3, and with assistance of the self-assembled FcFFRGD nanostructures, DOX can be uptaken more efficiently, as evidenced by the higher red fluorescence signals inside HeLa cells than that of free drug. These studies indicated that the FcFFRGD nanoparticle with a hydrophobic inner core and spatially arranged RGD ligands on outside surface can also be exploited for targeting intracellular drug delivery via αvβ3 integrin binding (Figure S12 and S13). 4. CONCLUSIONS In summary, we introduced here a new kind of peptide analogue, Fc-FFRGD, which contained a ferrocene group (Fc), a Phe-Phe dipeptide segment and a RGD ligand. Because of the amphiphilicity of its molecular structure and strong non-covalent interactions, this molecule exhibited good self-assembling ability to form stable nanostructures and hydrogel in water. More importantly, owing to the presence of ordered array of RGD ligands on the surface of nanostructures, the supramolecular assemblies (e.g., nanoparticle and hydrogel) of Fc-FFRGD exhibited high potential to work as a new biomimetic material for cell adhesion and growth or a nanocarrier for drug encapsulation and delivery via multiple integrin-dependent interactions.


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ASSOCIATED CONTENT Supporting Information. 1H NMR, mass spectrum and gel stability of Fc-FFRGD. Drug encapsulation and delivery cytotoxicity assay and flow cytometry analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Email: [email protected]; [email protected] ACKNOWLEDGMENT We thank Dr. Lin Guo for discussion and manuscript editing assistance. This work was supported financially by the National Program on Key Research Project of China (Ministry of Science and Technology of China, 2016YFC1100100); the National Natural Science Foundation of China (51673142) to X. Li, Scientific Research Foundation for the Returned Overseas Chinese Scholars (State Education Ministry of China); the Natural Science Foundation of Jiangsu Province (BK20151218, BK20140326) to X. Li and Y. Pan, respectively; the Natural Science Foundation of Jiangsu Province, China (BK20130290 ) to L. Guo; State Key Laboratory of Molecular Engineering of Polymers (Fudan University) to X. Li; the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). REFERENCES 1. Hynes, R. O., Integrins-Versatility, Modulation, and Signaling in Cell-Adhesion. Cell 1992, 69, 11-25. 2. Palecek, S. P.; Loftus, J. C.; Ginsberg, M. H.; Lauffenburger, D. A.; Horwitz, A. F., IntegrinLigand Binding Properties Govern Cell Migration Speed through Cell-Substratum Adhesiveness. Nature 1997, 385, 537-540. 3. Dunehoo, A. L.; Anderson, M.; Majumdar, S.; Kobayashi, N.; Berkland, C.; Siahaan, T. J., Cell Adhesion Molecules for Targeted Drug Delivery. J. Pharm. Sci. 2006, 95, 1856-1872.


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Scheme 1. Illustration of the self-assembling process of Fc-FFRGD for the generation of supramolecular nanofibers and hydrogel.


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Scheme 2. The synthetic route for the preparation of hydrogelator Fc-FFRGD.


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Figure 1. A) and B) Photographs of the sol-gel transition of Fc-FFRGD (1.0 wt%, pH=7.4); C) strain dependence and D) frequency dependence of the dynamic storage moduli (G’) and the loss moduli (G’’) of hydrogel Fc-FFRGD shown in Figure 1B.


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Figure 2. A-G) TEM and H-N) SEM images of the self-assembling nanostructures of FcFFRGD from metastable nanospheres to well-defined nanofibers in the course of 24 h.


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Figure 3. A) DLS analysis of the hydrodynamic radius and size distribution of nanostructures formed by Fc-FFRGD as shown in Figure 2; B) dynamic time sweep of the sample of FcFFRGD shown in Figure 1A to examine the transformation from solution to hydrogel with time; C) CD spectroscopy and D) FTIR analysis of the sample of Fc-FFRGD shown in Figure 1 to reveal the secondary structure transition with time.


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Figure 4. A) Fluorescence images of live/dead assays of NIH/3T3 fibroblast cells and human umbilical vein endothelial cells (HUVEC) cultured on hydrogel Fc-FFRGD (1.0 wt%, pH=7.4) over the course of 5 days; B) cell densities of NIH/3T3 cells and HUVECs determined by cell counting with a hemocytometer over the course of a 5-day culture on hydrogel Fc-FFRGD shown in Fig. 4A; cell spreading of C) NIH/3T3 cells and D) HUVECs on hydrogel Fc-FFRGD, in which actin filaments were stained with fluorescein-phalloidin and nuclei were with DAPI.


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Figure 5. Confocal fluorescence and bright field microscopy images of HeLa cells treated by free doxorubicin (10 µM) and Fc-FFRGD vesicles encapsulating doxorubicin (under the concentration of doxorubicin at 10 µM).


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Integration of a structural motif possessing self-assembling properties with a cell adhesive ligand (e.g., RGD) would offer an attractive method for preparing novel biomaterials with spatially orienting RGD ligands on their surfaces for integrin binding and cell-material recognition.


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Supramolecular Self-Assemblies with Nanoscale ... - ACS Publications

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