Luminescent LanthanideâCollagen Peptide Framework for pH

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A luminescent lanthanide-collagen peptide framework for pH-controlled drug delivery Linyan Yao, Yue Hu, Zhao Liu, Xiao Ding, Jing Tian, and Jianxi Xiao Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b01160 • Publication Date (Web): 28 Dec 2018 Downloaded from http://pubs.acs.org on January 1, 2019

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Molecular Pharmaceutics

A luminescent lanthanide-collagen peptide framework for pH-controlled drug delivery Linyan Yao, Yue Hu, Zhao Liu, Xiao Ding, Jing Tian, and Jianxi Xiao* State Key Laboratory of Applied Organic Chemistry, Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, PR China E-mail: [email protected]

Keywords: Collagen mimetic peptides, scaffold, self-assembly, pH-responsive, drug delivery

Abstract Collagen mimetic scaffolds play a pivotal role in regenerative medicine and tissue engineering due to their extraordinary structural and biological features. We have herein for the first time reported the construction of luminescent lanthanide-collagen peptide hybrid three-dimensional nanofibrous scaffolds, which well mimic the characteristic architectural structure of native collagen. Three collagen mimetic peptides composed of repetitive central (GPO)7 sequences and altered terminal amino acids, have been shown to consistently self-assemble to form biocompatible nanofibers under the trigger of a variety of lanthanide ions, which also functionalize the assembled materials with easily tunable photoluminescence. Furthermore, the collagen peptide-lanthanide hybrid scaffolds possess programmable pH-responsive features. The lanthanide ion-mediated assembly of all the three collagen peptides are conveniently and reversibly regulated by pH, while their pHdependent patterns are finely tuned by the identity of terminal amino acids. Using camptothecin and cefoperazone sodium as two model drugs, the drug-loading and releasing efficiency of the collagen peptide-lanthanide scaffolds are nicely modulated by pH, demonstrating the efficacy of these nanofibrous scaffolds as pH-responsive drug carriers. These novel luminescent collagen peptide-lanthanide scaffolds provide a facile

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system for pH-controlled drug delivery, suggesting promising applications in the development of therapies for many diseases.

1. Introduction Design of biomimetic scaffold plays a pivotal role in regenerative medicine and tissue engineering.1-3 A variety of biomaterials have been developed to fabricate three-dimensional scaffold, which provides an appropriate environment for cell attachment and drug release.4, 5 Synthetic polymers such as the aliphatic polyester family of biodegradable polymers, have received extensive attention for use in tissue engineering; however, their applications are often limited by biocompatibility concerns due to manufacturing residuals or degradation products.6-8 Natural biomaterials, including proteins, polysaccharides and glycoproteins, have thus all been tailored for scaffold materials thanks to their inherent superior biocompatibility.9-11 Collagen, the major component of extracellular matrix, has been an attractive candidate for a broad range of biomedical applications arising from its natural hierarchical structures and biological interactions.12-14 The distinct (Gly-X-Y)n amino acid sequence pattern leads collagen to form rod-like triple helices, which further self-assemble to form D-periodic fibrils.15,

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These fibrils are cross-linked to form a molecular scaffold, which provides

mechanical strength and structural integrity to connective tissues. Furthermore, the collagen triple helix interacts with a number of cell surface receptors to mediate cell adhesion, proliferation and migration, while its interaction with other extracellular matrix molecules regulates matrix structure and remodeling.17 Collagen-based scaffolds have shown wide applications in bladder and urethra repair, skin substitute, and facial soft tissue augmentation.18-21 However, there are increasing concerns of the use of animal-derived collagen regarding the transmission of pathogens.22 The use of recombinant system to produce


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collagen remains complicated concerning the specific proline hydroxylation in the Y-position of collagen sequences.23 These difficulties have spurred extensive interest to develop collagen mimetic peptides to form well-ordered supramolecular structures.24 A variety of approaches such as - stacking, -cation interactions, metal-ligand interactions, electrostatic interactions, hydrophobic interactions, peptide amphiphiles, and triple helical nucleation have been established to generate collagen fibers.25-34 Self-assembly of collagen mimetic peptides has also been contributed to the creation of other higher-order structures, including hollow spheres, nanodisks, nonosheets and microflorettes.35-40 A three-dimensional matrix has recently been constructed using terminally modified collagen mimetic peptides and Ni2+ as an external stimulus for cellular encapsulation.41 We herein report the construction of a lanthanide-collagen peptide framework for pHcontrolled drug delivery. Metal-organic frameworks (MOFs) and coordination polymers built by linking metal ions via strong covalent interactions, have been widely utilized to synthesize novel materials with targeted functionality.42, 43 The superior properties of MOFs, such as intrinsic biodegradability, tailorable composition and structure, high biocompatibility and versatile functionality, enable them as fabulous drug delivery carriers.44, 45 A variety of MOFs based on different types of metal ions have been developed for pH-responsive anticancer drug delivery.46,

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Particularly, lanthanide-organic frameworks (Ln-MOFs) have received

increasing attention in cancer diagnosis and therapy, owing to the extraordinary features of lanthanide ions such as high photochemical stability, tunable emissions and long luminescence lifetime.48, 49 We have for the first time synthesized biocompatible lanthanidecollagen peptide hybrid three-dimensional nanofibrous scaffold. We have demonstrated that the terminal amino acids of the constructed collagen peptides finely tuned the pH-sensitive features of the assembled luminescent scaffold, which provided promising platforms for pHcontrolled drug delivery. ACS Paragon Plus Environment


2. Experimental Section 2.1. Peptide synthesis Peptides (DDColDD, DWColWD, and HWColWD) were synthesized in-house by standard Fmoc solid phase synthesis method using 2-chlorotrityl chloride resin at a 0.1 mmol scale. Briefly, stepwise couplings of amino acids were achieved using a double coupling method with Fmoc-amino acids (4 eq.), DIEA ( 6 eq.) and activator reagents (HBTU + HOBt 0.66 mmol/ml, 4 eq.). The reaction mixture was washed with DMF (3 x 5 ml) and DCM (3 x 5 ml) following each step of coupling, and the Fmoc protection group was removed with 20% piperidine in DMF. Test reagent (2% ethanal DMF, 2% chloranil DMF) was employed to monitor the completion of each coupling reaction and Fmoc deprotetction. The peptides were obtained by treating the resin with TFA/TIS/H2O (95:2.5:2.5) for 3 hrs to remove the tBu groups and to cleave itself from the resin. Cold Et2O was used to precipitate the peptide. The precipitates were resuspended in cold Et2O, sonicated and centrifuged again. Crude products were then dissolved in water, and lyophilized. The peptides were purified using reverse phase HPLC on a C18 column. The purity of the peptides were confirmed by mass spectrometry. m/z calculated 2349.5 [M]+ for DDColDD, found 2348.0 [M]+; m/z calculated 2491.8 [M]+ for DWColWD, found 2491.1 [M]+; m/z calculated 2513.8 [M]+ for HWColWD, found 2513.1 [M]+.

2.2. Circular Dichroism Spectroscopy CD spectra were recorded on a Chirascan CD spectrometer (Applied Photophysics, UK) equipped with a Peltier temperature controller. The peptides with a concentration of 1.0 mg/mL were prepared in 20 mM acetic acid-sodium acetate buffer (pH 3.0 and pH 5.8), and in 20 mM Tris-HCl buffer (pH 7.0), respectively. The samples were equilibrated for at least 24 hrs at 4 °C prior to the CD measurements. Cells with a path length of 1 mm were used. ACS Paragon Plus Environment

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Wavelength scans were conducted from 215 to 260 nm with a 0.5 nm increment per step and a 0.5 sec averaging time. Thermal unfolding curves were measured by monitoring the amplitude of the characteristic CD peak at 225 nm, while the temperature was increased 0.4 °C/min from 4 °C to 80 °C with an equilibration time of 2 min at each temperature. The melting temperature (Tm) was derived from the first derivative of the thermal unfolding curves.

2.3. Scanning Electron Microscopy SEM images of the peptide-Ln3+ aggregates were acquired using a Hitachi S-4800 scanning electron microscope (Hitachi Limited, Japan) with an operating voltage of 5.0 kV. Peptides DDColDD, DWColWD and HWColWD with a concentration of 3 mg/mL were prepared in 100 mM HEPES buffer at pH 7.0, and was incubated with various lanthanide ions at a molar ratio of 1:1 at 4 °C for 24 hrs. Peptide DWColWD-La3+ mixtures were prepared under different conditions by varying the peptide concentration (1 mg/mL - 5 mg/mL), the molar ratio of peptide : La3+ (1:1 - 1:4), incubation temperature (4 °C - 20 °C), incubation time (12 hrs - 48 hrs). White precipitates were collected by centrifugation, washed with alcohol three times, and finally resuspended in alcohol. A droplet of the resuspended solution was air-dried on a silica slice, and the dried samples were sputter-coated with AuPd for 2 min prior to imaging.

2.4. Fluorescence Spectroscopy Fluorescence spectra were recorded on a Hitachi FLS920 spectrofluorometer (Edinburgh Instruments company) equipped with a 400 W Xe lamp. The peptide-Eu3+ / Tb3+ aggregates were harvested after incubation at 4 °C for 24 hrs. The emission spectra were acquired for the peptide-Eu3+ / Tb3+ aggregates at an excitation wavelength of 394 nm and 294 nm, respectively. Fluorescence measurements were conducted from 420 to 720 nm with a 1 nm ACS Paragon Plus Environment


increment per step. The dwell time was 0.20 s, and the scan slits were set at 0.5040 nm. All emission spectra were baseline-corrected.

2.5. Turbidity Measurements Turbidity experiments were conducted on a JASCO V-630 spectrophotometer by measuring the optical density at 313 nm. Solutions of peptides DDColDD, DWColWD and HWColWD with a concentration of 3 mg/mL were prepared in different buffers: 100 mM HEPES buffer (pH=6.8, 7.0), 100 mM MES buffer (pH=5.8, 6.0), and 200 mM acetic acid/sodium acetate buffer (pH=3.0, 3.5, 4.0, 4.5, 5.0, 5.5). The peptide was incubated with equimolar La3+ / Eu3+ ions at 4 °C for 24 hrs. Then 500 µL of the peptide-La3+ or peptide-Eu3+ solutions was diluted to 2.5 mL, and its turbidity was immediately measured.

2.6. Drug loading of peptide DDColDD-La3+ nanofiber scaffolds Camptothecin and cefoperazone sodium were used as two model drugs. Mixtures of 0.51 mM peptide DDColDD and 0.17 mM camptothecin were prepared under three pH conditions: pH 7.0, 100 mM HEPES buffer; pH 5.8, 100 mM MES buffer; pH 3.0, 200 mM acetic acid/sodium acetate buffer. Then 0.17 mM La3+ ion was added to the mixture, leading to immediate aggregation, in which camptothecin was entrapped. The assembled materials were collected by centrifugation and washed with alcohol two times. The supernatants were collected and subjected to the UV measurements on a JASCO V-630 spectrophotometer. The amount of camptothecin in the supernatants was determined in reference to a standard curve of free camptothecin using the UV absorption at 368 nm. Similarly, mixtures of 1.28 mM peptide DDColDD, 0.43 mM cefoperazone sodium and 0.43 mM La3+ ions were prepared under these three pH conditions. The assembled materials were collected by centrifugation, and the supernatants were subjected to the UV measurements on a JASCO V-630 spectrophotometer. The amount of cefoperazone sodium in ACS Paragon Plus Environment

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the supernatants was determined in reference to a standard curve of free cefoperazone sodium using the UV absorption at 270 nm. The amount of drug (camptothecin or cefoperazone sodium) loaded into the peptide DDColDD-La3+ nanofiber scaffolds was thus calculated as the amount of drug initially prepared in solution deducted by its corresponding amount in the supernatants.

2.7. In vitro drug release of peptide DDColDD-La3+ nanofiber scaffolds Drug Release experiments were conducted on a JASCO V-630 spectrophotometer at three pHs (pH 7.0, 5.8 and 3.0). Briefly, equal amounts of peptide-La3+ assembled materials were suspended in three different buffers: pH 7.0, 100 mM HEPES buffer; pH 5.8, 100 mM MES buffer; pH 3.0, 200 mM acetic acid/sodium acetate buffer. The solutions were incubated for 24 hrs, and the supernatants were collected by centrifugation. Camptothecin and cefoperazone sodium released in the supernatants were determined by measuring the UV absorption at 368 nm and 270 nm, respectively.

2.8. In vitro Cytotoxicity of collagen mimetic peptides In vitro cytotoxicity of collagen peptide-lanthanide scaffold was evaluated by examining the viability of HELA cells according to China National Standard (GB/T 16886.5-2003 / ISO 10993.5:1999). The HELA cells were cultured in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% (V/V) Hyclone fetal bovine serum (FBS) and 1% streptomycin and penicillin in a humidified atmosphere of 5% CO2 at 37 °C. Cells were placed in a 96-well cell-culture plate at a density of 5 × 103 cells / 100 μL cell culture medium in each well and incubated for 4 hrs to allow attachment. Then the solutions of peptidelanthanide nanomaterials were added into the 96-well plate with four different final concentrations (0, 0.025, 0.05, and 0.1 mg/mL). The same amount of DMEM was added in other wells as control groups to make both the experimental and control groups reach a final ACS Paragon Plus Environment


volume of 20 μL culture medium. The cell cultures were then kept incubated in the humidified atmosphere with 5% CO2 at 37 °C for 48 hrs. After the addition of 10 μL 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) into each well, the specimens were incubated for another 4 hrs at 37 °C in darkness. Then 100 μL of dimethylsulfoxide (DMSO) was added into each well and shaked for 10 minutes. The optical density at 490 nm was determined by ELx800 Absorbance Microplate Reader (Bio-Tek, Winooski, VT, USA). Cell viability was calculated as the mean absorption value of three measurements of each condition divided by the mean absorption value of the control group.

3. Results 3.1. Design of the collagen mimetic peptides All the peptides are mainly composed of the repetitive Gly-Pro-Hyp sequences, which are the most abundant triplets in native collagen and are known to be the most stabilizing sequences for triple helix structure (Figure 1a).50 Aspartic acids are added at both terminals of the peptides in order to facilitate the strong interaction between lanthanide ions and collagen mimetic peptides, which have been recently reported to trigger the assembly of helical nanoropes.51 The first peptide DDColDD (with amino acid sequence DD(GPO)7DD) contains two aspartic acids at each terminus as binding units for lanthanide ions. A second peptide DWColWD (with amino acid sequence DW(GPO)7WD) is designed by replacing the second aspartic acid with tryptophan at each terminus in order to evaluate if the identity of the second terminal amino acid affects the self-assembly. Trp is chosen since it has been reported to sensitize the luminescence of lanthanide ion Tb 3+ owing to the antenna effect.52, 53 It has been observed that collagen mimetic peptides with terminal histine residues can self-assemble to form well-ordered supramolecular structures under the trigger of divalent metal ions.39 Therefore, a third peptide HWColWD (with amino acid sequence HW(GPO)7WD) is constructed by replacing the N-terminal aspartic acid ACS Paragon Plus Environment

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with histidine in peptide DWColWD in order to investigate if His can function as another binding unit for lanthanide ions besides Asp. Notably, all the three peptides consist of only natural amino acids, therefore, they are highly biocompatible, and can be facilely synthesized without the need of extra modification by organic compounds. The terminal amino acids are supposed to assist the binding of the peptides with lanthanide ions, promoting the head-to-tail self-assembly of the inorganic-organic hybrid system into well-ordered suparmolecular structures (Figure 1b). The free N- and C-termini may be also involved in the binding with lanthanide ions, while the favorable positioning of the terminal His and Asp by the triple helix structure probably played a determinant role in mediating the binding of the peptides with lanthanide ions. Furthermore, different terminal amino acids are examined to evaluate how they could affect the pH-responsive features of the collagen peptide-lanthanide system.

3.2. Triple helix structure of the collagen mimetic peptides In order to ensure the proper positioning of the terminal amino acids for the covalent binding with lanthanide ions, the formation of triple helix structure of collagen mimetic peptides is required. Circular dichroism (CD) spectra of peptide DDColDD displayed a maximum absorption at 225 nm at pH 7 and pH 3, indicating the formation of triple helix structure characteristic of native collagen at both pHs (Figure S1). Thermal transition studies further showed that peptide DDColDD formed a stable triple helix with a melting temperature (Tm) of 35.0 °C at pH 7 and 38.5 °C at pH 3, respectively (Figure S1). It suggested that the inclusion of terminal charged aspartic acids did not prevent the peptide from adopting a triple helix structure, and they only slightly reduced the thermal stability of the peptide due to the electrical repulsion. Similar cases were observed for peptides DWColWD and HWColWD, and their CD spectra showed the characteristic peak of triple helix structure for both peptides (Figure S2-3). ACS Paragon Plus Environment


Thermal transition studies further showed that peptide DWColWD formed a stable triple helix with Tm of 33.1 °C at pH 7, 37.6 °C at pH 5.8, and 39.2 °C at pH 3, respectively; while peptide HWColWD possessed Tm of 34.0 °C at pH 7 and 35.2 °C at pH 3, respectively. These results indicated that all the three peptides maintained the distinct triple helix structure of collagen under a broad range of pH conditions, while the presence of terminal charged amino acids finely tuned the thermal stability of the peptides.

3.3. Lanthanide ion-triggered reversible self-assembly of the collagen mimetic peptides The capability of various lanthanide ions to trigger the self-assembly of the three peptides DDColDD, DWColWD and HWColWD was examined in 100 mM Hepes buffer, pH 7.0 at 4 °C. All the peptide solutions remained clear prior to the addition of lanthanide ions (Figure 1c). When any kind of lanthanide ions (La3+, Eu3+, Tb3+, Ce3+, Er3+, Tm3+, and Yb3+) was added to the peptide solution, it immediately became turbid, indicating that all the three peptides can be triggered by lanthanide ions to self-assemble (Figure 1c). When the commonly used divalent metal ions (Mg2+, Ca2+, Ba2+, Mn2+, Co2+, and Ni2+) were added to the peptide solutions, they all remained clear (data not shown). However, the addition of Fe2+ and Cu2+ ions led to turbid solutions for all the three peptides, while the addition of Zn2+ resulted in aggregates only for peptide HWColWD. These results suggested that the selfassembly of the three peptides was majorly specific to lanthanide ions, while the modification of terminal amino acids finely tuned their ability to coordinate with divalent metal ions. The reversibility of the assembly of the three peptides was evaluated by chelation competition experiments with ethylenediaminetetraacetic acid (EDTA). When excess EDTA was added to the turbid solution containing lanthanide ions, the aggregates disappeared and the solution became transparent within a minute (Figure 1c). These results indicated that the lanthanide ion-mediated assembly of the three peptides could be efficiently reversed by EDTA.


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Lanthanide ions are likely to play a crucial role in the readily reversible assembly of the constructed collagen mimetic peptides.

3.4. Morphology of self-assembled collagen peptide-lanthanide nanomaterials Scanning electron microscopy (SEM) was employed to investigate the morphology of the assembled materials of peptides DDColDD, DWColWD and HWColWD complexed with various lanthanide ions (Figure 2). SEM images of peptide DDColDD assemblies with La3+, Eu3+, and Tb3+ at a molar ratio of 1:1 all led to the formation of three-dimensional nanofibrous scaffolds (Figure 2a-c). Similarly, the assemblies of peptides DWColWD and HWColWD triggered by lanthanide ions (La3+, Eu3+, and Tb3+) all revealed interconnected nanofibrous supramolecular structures (Figure 2d-i). It demonstrated that the type of lanthanide ions have little effect on the morphology of the peptide assemblies. SEM images of peptides DDColDD, DWColWD and HWColWD complexed with divalent metal ions Fe2+ and Cu2+ did not show any well-ordered nanostructures (Figure S4). A control peptide EEColEE (with amino acid sequence EE(GPO)7EE) was constructed to evaluate the role of Glu and Asp as terminal amino acids in the coordination with lanthanide ions. SEM images of peptide EEColEE-La3+ assemblies indicated that peptide EEColEE could selfassemble to form nanofibrous scaffolds under the trigger of lanthanide ions, suggesting that the inclusion of Glu at the terminals of collagen peptides shared similar capability as Asp to coordinate with lanthanide ions (Figure S5). In conclusion, the constructed three peptides were all specifically mediated by lanthanide ions to assemble to form nanofibers, and all the types of tested lanthanide ions displayed similar capability to control the selfassembly of the peptides. The assembly of DWColWD-La3+ further investigated in more details considering the concentration of peptide DWColWD (Figure S6). Peptide DWColWD with various concentrations (1 mg/ml, 3 mg/ml, and 5 mg/ml) were examined, and the addition of ACS Paragon Plus Environment


equimolar La3+ always produced nanofibrous scaffolds at 4 °C. Well-ordered nanofibrous structures were also consistently achieved while varying the molar ratios of La3+: DW(GPO)7WD (1:1, 2:1, and 4:1), the incubation temperatures (4°C, 15°C, and 20°C) and the incubation time (12, 24, and 48 hrs) for the DWColWD-La3+ mixture (Figure S7-S9). In vitro cytotoxicity of collagen peptide-La3+ scaffolds were evaluated by examining the viability of rabbit bone mesenchymal stem cells (Figure S10). All the scaffolds (DDColDDLa3+, DWColWD-La3+ and HWColWD-La3+) displayed similarly high cell viability at various concentrations, demonstrating that the collagen peptide-La3+ scaffolds were highly biocompatible. All these results demonstrated that our constructed collagen peptide-lanthanide ion system provided a simple and reliable approach for steadily generating biocompatible well-ordered assemblies with nanofibrous scaffold structures.

3.5. Photoluminescence of the assembled collagen peptide-lanthanide nanomaterials The photoluminescent features of the assembled nanomaterials of peptides DDColDD, DWColWD and HWColWD with Eu3+ and Tb3+ were evaluated by solid state fluorescence (Figure 3). Eu3+ and Tb3+ were selected as two typical luminescent lanthanide ions, since La3+ didn’t possess any luminescent features. The luminescence spectrum of the DDColDD-Eu3+ assembled materials possessed multiple peaks assigned to the intra-4f6 5D0-7F0-2 transitions at an excitation wavelength of 394 nm (Figure 3a). The DDColDD-Eu3+ assembled materials were red-light emitters under UV light irradiation (394 nm), and the presence of a strong peak for 5D0-7F2 suggested that the DDColDD-Eu3+ assemblies displayed good color purity. The assembled nanomaterials for peptides DWColWD and HWColWD complexed with Eu3+ ions showed similar luminescence spectra (Figure 3b-c). The luminescence spectrum of the DDColDD-Tb3+ assembled materials displayed multiple peaks assigned for the intra-4f8 5D4-7F6-3 transitions at an excitation wavelength of 294 nm (Figure 3d). The DDColDD-Tb3+ assembled materials were green-light emitters under UV ACS Paragon Plus Environment

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light irradiation (294 nm), and the strong peak of 5D4-7F5 demonstrated that the DDColDDTb3+ assembled materials displayed excellent color purity. The assembled nanomaterials for peptides DWColWD and HWColWD complexed with Tb3+ ions showed similar luminescence spectra (Figure 3e-f). Lanthanide ions are well known to emit across the entire visible spectrum (red-Eu3+, Pr3+, Sm3+; green-Tb3+, Er3+; blue-Tm3+, Ce3+, Dy3+) with high color purity.54 Our results demonstrated that the assembled nanomaterials for all the three peptides complexed with lanthanide ions displayed good photoluminescent features, and their colors could be conveniently modulated by utilizing different types of lanthanide ions.

3.6. pH-responsive self-assembly of the collagen mimetic peptides The lanthanide ion-triggered assembly performance of peptides DDColDD, DWColWD and HWColWD was examined under various pH conditions by turbidity measurements. The collagen peptide-La3+ solutions were all turbid at pH 7 and 5.8, while the aggregates became disappeared when pH was lowered to 3.0 (Figure 4a). SEM images of collagen peptide-La3+ assemblies at pH 5.8 indicated that they all formed well-ordered nanofibrous scaffolds, which shared the same structural features as the collagen peptide-La3+ assemblies prepared at pH 7.0 (Figure S11). The pH-dependence of the peptide-La3+ assembly was further investigated by monitoring the optical density at 313 nm (Figure 4b). For peptide DDColDD, when pH was decreased from 7.0 to 3.0, its OD 313nm became reduced, and reached the minimum at pH 5.5. For peptides DWColWD and HWColWD, their OD313nm values were decreased with pH differently, and they reached a plateau around pH 4.0. It revealed that the pH-dependence of the peptide-La3+ assembly may be finely tuned by the identity of terminal amino acids, and it was probably a combined effect of Trp, Asp and His on the coordination capability of La3+. Similar phenomenon were also observed for the peptideEu3+ assemblies, suggesting that all the constructed peptide-lanthanide assemblies were pH-dependent (Figure S12). ACS Paragon Plus Environment


The reversibility of the pH-mediated peptide-lanthanide assembly was also evaluated (Figure 5). When pH of the peptide DWColWD-La3+ solution was adjusted from pH 7.0 to pH 3.0 by the dropwise addition of hydrochloric acid, the mixture immediately became clear (Figure 5a-b). When pH of the solution was adapted back from pH 3.0 to pH 7.0 by the dropwise addition of sodium hydroxide, it immediately became turbid again (Figure 5b-c). SEM visualization indicated that the regenerated aggregates appeared to display similar nanofibrous structure as the original aggregates prepared at pH 7, indicating that the peptideLa3+ assembly was facilely and reversibly regulated by pH. Similar cases were also observed for the DWColWD-Eu3+ assembly, suggesting that all the constructed peptidelanthanide ion assemblies were pH-adjusted reversible (Figure S13).

3.7. Drug loading and in-vitro drug release of peptide-La3+ nanofibrous scaffolds The pH-responsive properties of the collagen mimetic peptide-lanthanide scaffolds were investigated for the potential use as drug carriers. Camptothecin was widely used in the investigation of delivery effectiveness of anticancer drugs, and was applied here as a model drug.55, 56 DDColDD was chosen as the example peptide. Mixtures of peptide DDColDD and camptothecin were prepared under three pH conditions: pH 7.0, 5.8, and 3.0. The addition of La3+ ions in the mixtures at pH 7.0 and pH 5.8 immediately generated aggregates, in which camptothecin was entrapped. Supernatant was then collected by removing the aggregates and the content of camptothecin was determined by UV. The amount of drug loaded into the peptide DDColDD-La3+ scaffold was thus calculated as the amount of camptothecin initially prepared in solution deducted by its corresponding amount in the supernatant. The drug camptothecin showed a strong absorption at 368 nm, while the peptide or La3+ ions displayed little absorption (Figure 6a). The solution of camptothecin together with the peptide or La3+ ions showed similar UV profiles as the drug alone, indicating that the peptide or La3+ ions would not interfere in the UV ACS Paragon Plus Environment

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measurements of camptothecin. UV profiles of drug camptothecin incubated with La3+ at different molar ratios in various buffers were almost completely overlapped, suggesting that there was no significant interaction between camptothecin and La3+ ions (Figure S14). When compared with the original drug added in the solution, the UV absorption of camptothecin in the supernatant became significantly decreased, indicating the successful entrapment of camptothecin in the DDColDD-La3+ scaffold at pH 7.0 (Figure 6a). The loading efficiency of camptothecin by the DDColDD-La3+ scaffold at pH 7.0 was calculated as 47.4 mg/g using the standard UV curve of camptothecin. Similar UV measurements were also performed at pH 5.8 (Figure 6b). When compared with the original drug added in the solution, the UV absorption of camptothecin in the supernatant became significantly decreased, indicating the successful entrapment of camptothecin in the DDColDD-La3+ scaffold at pH 5.8. The amount of drug loaded into the DDColDD-La3+ scaffold was calculated as 42.5 mg/g, which was less than the drug loaded at pH 7.0. In contrast, UV profiles of camptothecin in the supernatant was the same as the original drug added in the solution, since no aggregates were formed, and therefore no drug was loaded at pH 3.0 (Figure 6c). These results demonstrated that the DDColDD-La3+ scaffolds could be used as drug carriers, while their drug loading efficiency was mediated by pH and the optimal loading was achieved at pH 7.0. Drug release experiments were conducted for the drug-loaded DDColDD-La3+ scaffolds under three pH conditions (pH 7.0, 5.8 and 3.0). DDColDD-La3+-drug assembled materials were produced at pH 7.0, and an equal amount of the assembled nanomaterials were suspended in three different buffers: pH 7.0, 100 mM HEPES buffer; pH 5.8, 100 mM MES buffer; pH 3.0, 200 mM acetic acid/sodium acetate buffer. Camptothecin released in the supernatant was determined by measuring the UV absorption at 368 nm (Figure 6d). It indicated that camptothecin was most released at pH ACS Paragon Plus Environment


3.0 (34.3 mg/g), moderately released at pH 5.8 (10.9 mg/g), and least released at pH 7.0 (4.2 mg/g). The releasing efficiency of camptothecin was mediated by acidity, indicating that the DDColDD-La3+ scaffolds could be applied as drug carriers in a pH-responsive manner. Cefoperazone sodium, a popular antibiotic, was tested as another model drug.57 The drug cefoperazone sodium showed a strong absorption at 270 nm (Figure S15). The solution of cefoperazone sodium together with the peptide or La3+ ions showed similar UV profiles as the drug alone, indicating that the peptide or La3+ ions would not interfere in the UV measurements of cefoperazone sodium. The same UV profiles of drug cefoperazone sodium incubated with La3+ at different molar ratios suggested that there was no significant interaction between cefoperazone and La3+ ions (Figure S16). When compared with the original drug added in the solution, the UV absorption of cefoperazone in the supernatant became significantly decreased, indicating the successful entrapment of cefoperazone in the DDColDD-La3+ scaffold at pH 7.0 and pH 5.8 (Figure S15a-b). The loading efficiency of cefoperazone by the DDColDD-La3+ scaffold at pH 7.0 and pH 5.8 was calculated as 136.4 mg/g and 47.4 mg/g, respectively, using the standard UV curve of cefoperazone sodium. These results demonstrated that the loading efficiency of cefoperazone using the DDColDD-La3+ scaffold was mediated by pH and the optimal loading was achieved at pH 7.0. Drug release experiments showed that cefoperazone was most released at pH 3.0 (111.3 mg/g), moderately released at pH 5.8 (56.7 mg/g), and least released at pH 7.0 (40.2 mg/g), suggesting that the releasing efficiency of cefoperazone by the DDColDD-La3+ scaffold was modulated by acidity (Figure S15d). These results indicated that a wide variety of drugs could be loaded using the collagen peptide-lanthanide scaffolds, while the loading efficiency may be dependent on the identity of the drug. Hydrophilic drugs may be easier to be loaded or entrapped in the collagen peptide-lanthanide scaffolds. The drug loading ACS Paragon Plus Environment

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and releasing results of both camptothecin and cefoperazone indicated that the nanofibrous scaffolds of our constructed collagen mimetic peptide-lanthanide system provided an efficient pH-responsive drug carrier.

4. Discussion and Conclusions Due to the extraordinary structural and biological features, collagen-based scaffolds play an essential role in regenerative medicine and tissue engineering.12 However, the use of animalderived collagen has aroused concerns due to the potential risk of transmission of diseases, while the recombinant technology to produce hydroxyproline-containing collagen remains challenging.22, 23 Therefore, design of peptide-based nanomaterials, which can form scaffold structure and retain the functional properties of native collagen at the meantime, is of pivotal importance to build improved functional biomaterials. We have herein for the first time successfully created luminescent collagen peptidelanthanide ion hybrid three-dimensional scaffolds. Previous studies have shown that collagen mimetic peptides modified with three metal binding units, a nitrilotriacetic acid (NTA) unit at the N terminus, a His2 sequence at the C terminus, and a bipyridyl (bipy) moiety at a central position, were capable to form scaffold structure triggered by transition metal ions.41 Compared with transition metal ions, lanthanide ions possess distinct fluorescent, magnetic and chemical features owing to its 4f electronic configuration, leading to broad applications of lanthanide-based nanomaterials in bioimaging, biosensing, drug delivery and phtodynamic therapy.58-60 Lanthanide ions not only play as an external stimulus to mediate the assembly, but also functionalize the assembled materials with fabulous properties such as easily tunable photoluminescence. Furthermore, compared with previous divalent metal-binding collagen mimetic peptides, our constructed lanthanide-binding peptides consist of only natural amino acids, which facilitates facile synthesis and high biocompatibility. ACS Paragon Plus Environment


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The constructed collagen peptide-lanthanide system provides a simple and robust approach to create self-assembled nanofibrous scaffolds, which well mimic the architectural structure of native collagen. We have recently reported the construction of a collagen mimetic peptide DD(PPG)12DD self-assembled to form helical nanoropes triggered by lanthanide ions.51 Herein, the inclusion of (GPO)7 instead of (PPG)12 sequences in the center of collagen mimetic peptides completely shifted the morphology of the peptide assemblies from nanoropes to nanofibers. It suggested that the GPO sequences may possess stronger capability to form fibrils than the GPP sequences, which was consistent with previous reports that the inclusion of hydroxyproline was essential for the fibril formation of collagen mimetic peptides.61 Previous studies have shown that other factors such as the location and number of metal chelation sites may also play a critical role in modulating the morphology of the peptide-metal assemblies.32, 40, 41 All the three peptides ((DDColDD, DWColWD and HWColWD) consist of (GPO)7 sequences in the middle, and they consistently form well-ordered nanofibrous structures under the trigger of a variety of lanthanide ions. It indicates that other featured amino acids besides Asp could be included as terminal amino acids for the construction of novel highly specific lanthanide

ion-binding

peptides.

All

the

constructed scaffolds

(DDColDD-La3+,

DWColWD-La3+ and HWColWD-La3+) are highly biocompatible, suggesting that the collagen peptide-lanthanide system is a reliable strategy for generating biocompatible wellordered assemblies with nanofibrous scaffold structures. Most notably, the collagen peptide-lanthanide nanofibrous scaffolds possess programmable pH-responsive features. The lanthanide ion-mediated assembly of all the three collagen peptides has been demonstrated to be conveniently and reversibly regulated by pH, while their pH-dependent patterns could be finely tuned by the identity of terminal amino acids. Using camptothecin and cefoperazone sodium as model drugs, the drug-loading and releasing efficiency of the collagen peptide-lanthanide scaffolds has ACS Paragon Plus Environment

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been shown to be nicely modulated by pH, demonstrating the efficacy of these nanofibrous scaffolds as pH-responsive drug carriers. It has long been established that a low pH condition is a hallmark of many diseases such as cancer, and the tumor-specific pH gradient has been extensively exploited for its treatment.62-64 The novel luminescent pH-responsive collagen peptide-lanthanide ion scaffolds provide a facile system for pHcontrolled drug delivery, and may have great potential in the development of therapies for these diseases.

Supporting Information Supporting Information is available.

Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (Grant No. 21775059) and the Fundamental Research Funds for the Central Universities (Grant No. lzujbky-2016-k10).

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Figure legends Figure 1. Lanthanide ion-triggered assembly of collagen mimetic peptides. (a) Amino acid sequences of three collagen mimetic peptides (DDColDD, DWColWD, and HWColWD) containing lanthanide ion-binding ligands at each terminus. (b) Schematic illustration of the assembly of the triple helical peptide upon the addition of lanthanide ions. (c) Photographs showing the visual changes of the peptide solution after the addition of lanthanide ions and EDTA.

Figure 2. SEM images of peptides DDColDD (a-c), DWColWD (d-f), and HWColWD (g-i) with specified lanthanide ions at a molar ratio of 1:1: La3+ (a, d, g), Eu3+ (b、e、h), and Tb3+ (c、f、i). Scale bar = 200 nm.

Figure 3. Fluorescence emission spectra of luminescent materials of peptides DDColDD (a, d), DWColWD (b, e), and HWColWD (c, f) with specified lanthanides: Eu3+ (a-c) and Tb3+ (d-f). The emission spectra were taken at an excitation wavelength of 394 nm and 294 nm for Eu3+ and Tb3+, respectively.

Figure 4. pH dependence of the peptide-La3+ assembly. Photographs show the visual changes of the peptide solution (DDColDD (a1-a3), DWColWD (b1-b3), and HWColWD (c1-c3)) after the addition of La3+ ions under different pH conditions: pH 7.0 (a1-c1), pH 5.8 (a2-c2), and pH 3.0 (a3-c3). Turbidity profiles of the peptide-La3+ mixtures in a broad range of pHs from 3.0 to 7.0 for the three peptides: DDColDD (black), DWColWD (blue), and HWColWD (red).

Figure 5. The pH-mediated reversibility of peptide-La3+ assembly. Photographs show the visual changes of the peptide-La3+ solution modulated from pH 7.0 (a) to pH 3.0 (b) by the addition of extra acids as well as the reverse process from pH 3.0 (b) back to pH 7.0 (c) by the ACS Paragon Plus Environment

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addition of extra sodium hydroxide. SEM images of the peptide-La3+ aggregates produced at the beginning and final stages at pH 7.0 were obtained (a, c). The molar ratio of peptide DWColWD: La3+ is 1:1. Scale bar =500 nm.

Figure 6. Drug loading and release of camptothecin using peptide DDColDD-La3+ nanofibrous scaffolds. UV profiles were measured for the solutions of drug camptothecin (black), peptide (red), La3+ (blue), the drug-peptide mixture (purple), the drug-La3+ mixture (magenta), and the supernatant after removing the aggregates in the peptidedrug-La3+ mixture (olive) under different pH conditions: pH 7.0 (a), pH=5.8 (b), and pH=3.0 (c). UV profiles of camptothecin released in the supernatants were measured after 24 hrs incubation of the drug-loaded peptide-La3+ scaffolds in three different buffers: 100 mM HEPES buffer pH 7.0 (black), 100 mM MES buffer pH 5.8 (red), and 200 mM acetic acid/sodium acetate buffer pH 3.0 (blue) (d).



Figure 1


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Figure 2



Figure 3


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Figure 4



Figure 5


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Figure 6



Figure 1


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Figure 2



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Luminescent LanthanideâCollagen Peptide Framework for pH

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