Luminescent Biofunctional Collagen Mimetic Nanofibers.

Collagen has long been one of the top targets for biomimetic design due to its superior structural and functional properties. Significant progress has been achieved to construct self-assembling peptides to mimic the fibrous nanostructure of native collagen, while it is still very demanding to fabricate peptide assemblies that can recapitulate both structural and biofunctional features of collagen. Herein, collagen-like peptides have been synthesized to contain negatively charged amino acids as the binding groups of lanthanide ions and the integrin-binding motif GFOGER. The simultaneous inclusion of negatively charged amino acids in the middle as well as at both terminals drives the peptides to self-assemble to form well-ordered nanofibers with distinct periodic banding patterns specifically mediated by lanthanide ions. The aggregation tendency and the morphology of the final assembled materials for the peptides are modulated in a pH-cooperative manner, which well mimics the pH-dependent fibrillogenesis of Type I collagen. The utilization of lanthanide ions in the system not only offers a convenient external stimulus but also functionalizes assembled materials with excellent luminescent features. Most notably, the lanthanide-triggered peptide assembled nanomaterials possess good cell adhesion properties, which resemble the biological function of collagen. This peptide-Ln3+ system provides a facile and potent strategy to generate nanofibers that mimic both the structural and functional properties of natural collagen. These novel pH-responsive, luminescent, and biofunctional collagen mimetic nanofibers open fascinating opportunities in the development of improved functional biomaterials in tissue engineering, drug delivery, and medical diagnostics.

Introduction

Collagen is a ubiquitous structural protein in mammals, and plays essential roles in the formation of tendon, bone, skin, cartilage, and ligaments.[1] Twenty-eight types of collagens have been discovered, and they possess a common triple helix structure.[2] The tight packing of the three α chains can only tolerate the least bulkiest Gly as every third residue, leading to the repetitive (Gly-X–Y)n amino acid sequence pattern.[3] The residues at the X and Y positions are frequently occupied by proline (Pro) and hydroxyproline (Hyp), respectively, with the latter enhancing the triple helix stability through water-bridged hydrogen bonds.[4] The signature triple helices assemble to form collagen fibrils, which provide a structural scaffold for the human body.[5] The triple helix structure is believed to be essential in collagen fibrillogenesis, and it is observed that the collagen fibrillogenesis boosts the stability of the triple helix.[6]

Collagen plays critical roles not only as structural components but also as functional molecules.[7] Collagen fibers are involved in the regulation of cell proliferation and migration, as well as extracellular matrix remodeling, through the interaction with a plethora of cell surface receptors and matrix biomolecules.[8,9] A number of cell-binding motifs, such as GFOGER, have been determined in collagen.[10−12] The specific binding of integrin with these bioactive motifs in collagen can activate cytoplasmic intracellular signaling pathways, which are considered to be critical for various biological processes such as embryogenesis, homeostasis, and tissue remodeling.[13−16] Furthermore, collagen fibers have displayed broad biomedical applications, such as skin substitute and facial soft tissue augmentation.[17,18] However, the utilization of animal collagen raises growing concerns due to the transmission of pathogens.[19] The development of biomimetic strategies to achieve superior structural and functional properties of collagen has therefore received intensive attention.[20]

A variety of triple-helical peptides have been constructed to form a plethora of well-ordered supramolecular structures such as fibers, microtubes, hollow spheres, nanodisks, nanosheets, nanoropes, nanowires, meshes, and microflorettes.[21−32] Most notably, extensive efforts have been contributed to the generation of fibrous nanomaterials, which best mimic the natural form of the most abundant type I collagen. Various approaches using modified cysteine-knots, π–π stacking, π-cation interactions, metal–ligand interactions, metal-mediated tandem coassembly, hydrophobic interactions, electrostatic interactions, triple-helical nucleation, peptide tessellation, and amphiphilic peptides have thus far been successfully established to achieve collagen fibers.[33−44] Among these collagen mimetic peptides, a few of their assembled nanomaterials display specific functions for nanowires, three-dimensional cell culture, platelet activation, and hydrolytic catalysis.[27,33,35,45−47] Particularly, a collagen mimetic peptide amphiphile containing the integrin-binding motif has recently been synthesized, which is capable of self-assembling to form nanofibers and to promote cell adhesion.[41] Up to now, significant progress has been made to fabricate self-assembling peptides to mimic the complex structure of native collagen, while it remains much more demanding to build collagen mimetic fibers with both structural and biofunctional features.[21,41]

We have recently established a collagen mimic peptide-lanthanide ion platform to form nanoropes via head-to-tail self-assembly through the specific binding between the terminal aspartic acids of the peptides and lanthanide ions.[22] We herein report the design of novel collagen mimetic peptides with negatively charged amino acids in the middle in addition to those at N- and C-terminals, which facilitate both radial and head-to-tail self-assembly of the peptides, leading to the formation of exquisite nanofibers characteristic of native collagen. Moreover, the inclusion of the integrin-binding motif GFOGER in these peptides empowers them with excellent cell adhesion features. The peptide-lanthanide system provides a robust tool to generate luminescent biofunctional collagen mimetic nanofibers. The synthesis of peptide-based nanomaterials, which well mimic both the structure and biological function of native collagen, has great potential in tissue regeneration, drug delivery, and medical diagnostics.

Results and Discussion

Design of Collagen Mimetic Peptides

We herein report the design of three collagen-like peptides that can self-assemble to form distinct fibers triggered by the addition of lanthanide ions. The self-assembling properties of the designed peptides CMP1 (with amino acid sequence DD(GPP)5GDP(GPP)6DD), CMP2 (with amino acid sequence DD(GPO)3GDOGPOGFOGERGPOGDO(GPO)3DD), and CMP3 (with amino acid sequence EE(GPO)3GEOGPOGFOGERGPOGEO(GPO)3EE) rely on the inclusion of negatively charged amino acids in the middle as well as at the terminals as the binding ligands for Ln3+ ions (Table S1). We have recently discovered that a triple-helical peptide DcolD with the amino acid sequence DD(PPG)12DD can be specifically mediated by Ln3+ ions to self-assemble to form banded helical nanoropes.[22] Aspartic acids have been demonstrated as excellent binding units for lanthanide ions, and their presence at both N- and C-terminals can mediate the head-to-tail self-assembly.[22]

We propose that the incorporation of extra aspartic acid in the middle of peptide CMP1 can facilitate the radial self-assembly, while the terminal aspartic acids promote the head-to-tail assembly, which will together drive the formation of nanofibers (Figure a,b). The (GPP)5 and (GPP)6 sequences besides GDP are included to reinforce high stability of the triple helix. The second peptide CMP2 adds the integrin-binding motif GFOGER, which has been demonstrated to functionalize triple-helical peptides with cell adhesion properties.[12,41] The GPO triplet is the most stable sequence for triple helix conformation and is used to enhance the triple helix stability of peptide CMP2.[48] The third peptide CMP3 replaces all aspartic acids of peptide CMP2 with glutamic acids to evaluate if glutamic acids share the same capability as aspartic acids to mediate the assembly. The three peptides are designed to self-assemble under the mediation of lanthanide ions to form nanofibers with structural and biofunctional features characteristic of native collagen.

Figure 1

Ln3+-triggered self-assembly of collagen mimetic peptides. (a) Amino acid sequences of three collagen-like peptides (CMP1, CMP2, and CMP3) containing Ln3+-binding ligands in the center as well as at the two terminals. (b) Schematic representation of the head-to-tail and radial assembly of the peptides triggered by lanthanide ions. (c) Photographs show the visual changes of the three peptide solutions before and after the addition of Ln3+ ions and ethylenediaminetetraacetic acid (EDTA).

Triple Helix Stability of Collagen Mimetic Peptides

The folding of collagen mimetic peptides into a triple helix structure is considered as a prerequisite to allow proper positioning of its carboxyl groups for binding with Ln3+ ions. Circular dichroism (CD) spectra of peptide CMP1 showed a characteristic peak at 225 nm, demonstrating the formation of triple helix structure (Figure S1). Thermal transition studies indicated that the melting temperature (Tm) of peptide CMP1 was 37 °C at pH 7.0 in 20 mM Tris-HCl buffer (Figure S1b,c). Similarly, CD spectra of peptides CMP2 and CMP3 displayed a typical peak of the triple helix structure at pH 7.0 (Figures S2 and 3). Thermal transition studies revealed that the melting temperature of peptide CMP2 and CMP3 was 43 and 48 °C, respectively. These results demonstrated that all of the three peptides possessed the unique triple helix structure of collagen.

Lanthanide-Mediated Assembly of Collagen Mimetic Peptides

The ability of lanthanide ions to initiate the assembly of peptide CMP1 was monitored in 100 mM Hepes buffer, pH 7.0 at 25 °C (Figure c). The peptide solution was transparent, and it immediately turned turbid after the addition of any type of trivalent lanthanide ions (La3+, Eu3+, Gd3+, Tb3+, Dy3+, Er3+, and Tm3+) (Figure c). In contrast, when the monovalent metal ions (Li+, Na+, K+, and Ag+) or divalent metal ions (Zn2+, Fe2+, Ni2+, Mg2+, Ca2+, Co2+, and Mn2+) were added to the peptide solution, it remained clear (data not shown). These results demonstrated that the self-assembly of peptide CMP1 was mainly specific to lanthanide ions. The reversibility of the peptide assembly was further examined by the addition of a metal chelator (Figure c). When EDTA (2.85 mM) was added to the turbid peptide CMP1-Ln3+ solution, it immediately became transparent, suggesting that EDTA could efficiently reverse the Ln3+-mediated assembly of peptide CMP1 (Figure c). These results revealed that the assembly of peptide CMP1 was conveniently regulated by lanthanide ions.

Similar to peptide CMP1, CMP2 and CMP3 were capable of self-assembling specifically under the trigger of lanthanide ions (Figure c). The addition of any type of trivalent lanthanide ions (La3+, Eu3+, Gd3+, Tb3+, Dy3+, Er3+, and Tm3+) turned the solution of CMP2 and CMP3 cloudy, while the turbid solutions of CMP2-Ln3+ or CMP3-Ln3+ immediately became clear after the addition of EDTA (Figure c). Meanwhile, the peptide solutions of CMP2 and CMP3 remained transparent after the addition of monovalent metal ions (Li+, Na+, K+, and Ag+) or divalent metal ions (Zn2+, Fe2+, Ni2+, Mg2+, Ca2+, Co2+, and Mn2+) (data not shown). These results demonstrated that lanthanide ions played the same critical role in the assembly of all of the three collagen-like peptides.

Morphology of Self-assembled Peptide CMP1-Lanthanide Nanomaterials

The morphology of the assembled nanomaterials of peptide CMP1 with different lanthanide ions was evaluated by scanning electron microscopy (SEM) (Figure ). The SEM image of the CMP1-La3+ aggregates at a molar ratio of 1:2 showed long and thin nanofibers (Figure a). Similarly, the peptide assemblies initiated by other lanthanide ions (Eu3+, Tb3+, and Dy3+) all showed cross-linked nanofibrous supramolecular structures (Figure b–d). The assembly of CMP1-La3+ was then examined under different conditions, such as the molar ratio of CMP1/La3+ and incubation time (Figure S4–5). A variety of relative concentrations of CMP1 and La3+ (1:1, 1:2, and 1:4) all resulted in well-ordered nanofibers (Figure S4). Nanofibers were also obtained using different incubation times (12, 24, and 48 h) for the CMP1-La3+ mixture (Figure S5). All of these results demonstrated that the CMP1-Ln3+ mixtures steadily led to delicate fibrous nanostructures.

Figure 2

Morphology of peptide CMP1-lanthanide nanomaterials. SEM images of peptide CMP1 with specified lanthanide ions La3+ (a), Eu3+ (b), Tb3+ (c), and Dy3+ (d) at a molar ratio of 1:2. Scale bar = 1.0 μm. Transmission electron microscopy (TEM) images of peptide CMP1 with La3+ ions at a molar ratio of 1:2 (e, f). Peptide CMP1-lanthanide nanomaterials were prepared in 100 mM Hepes buffer, pH 7.0.

Transmission electron microscopy (TEM) was employed to further characterize the structural details of the CMP1-La3+ assemblies at a nanometer scale (Figure e,f). TEM visualization of the La3+-triggered peptide assemblies showed an interconnected three-dimensional fibrous network (Figure e). Furthermore, the assemblies displayed distinct, regularly spaced bands, demonstrating the success of our design to be capable of imitating the characteristic banding feature of native collagen (Figure f). More notably, the distance between the banding gaps for the peptide-La3+ aggregates was estimated to be ∼10 nm (Figure f). This was in perfect match with the calculated length of peptide CMP1, suggesting that lanthanide ions triggered linear as well as radial assembly of the peptide (Figure b). These results convincingly demonstrated that the CMP1-La3+ system provided an excellent biomimetic approach to achieve the periodic banding of native collagen at the nanometer scale.

pH-Dependent CMP1-La3+ Assembly

The pH-responsive properties of the CMP1-La3+ system were then investigated under different pH conditions by turbidity and SEM (Figure ). When the pH of the mixture was adjusted from 7.0 to 3.5, a decreased amount of white aggregates was clearly observed, while the further decrease of pH to 3.2 and lower led to no visible aggregates (Figure ). It demonstrated that the La3+-triggered assembly of peptide CMP1 was significantly modulated by pH, which likely resulted from the pH-sensitive binding capability of aspartic acids and lanthanide ions. This constructed peptide-Ln3+ system well mimics the pH-dependent fibrillogenesis of Type I collagen, which self-assembles to form fibrils at pH 7.0, but not at acidic pH 2.5.[49]

Figure 3

pH dependence of the peptide CMP1-La3+ assembly. Photographs show the changes of the peptide solution after the addition of La3+ ions under various pH conditions (pH 7.0–2.6). SEM images of the CMP1-La3+ assembled materials prepared at different pH values: pH 7.0 (a), pH 6.4 (b), pH 6.0 (c), pH 5.0 (d), pH 4.0 (e), and pH 3.5 (f). The molar ratio of CMP1/La3+ is 1:2. Scale bar = 1.0 μm.

The morphology of the assembled materials at different pH values was characterized by SEM (Figure a–f). When the peptide solution became more acidic, the long and thin nanofibers became disrupted and less ordered. When the pH was decreased to 4.0 and 3.5, the peptide CMP1-La3+ aggregates turned to be very poorly organized, and the brick-like rather than fibrous suprastructures became dominant. These results indicated that pH not only controlled the extent of the peptide CMP1-Ln3+ assembly but also the morphology of the final assembled materials. The assembled materials of native Type I collagen displayed nice banded fibrils at pH 7.0, but much less ordered suprastructure at acidic pH, demonstrating the superior capability of this peptide-Ln3+ system to recapitulate the pH-responsive feature of native collagen.[49]

The reversibility of the pH-regulated peptide CMP1-Ln3+ assembly was further examined (Figure S6). The CMP1-La3+ mixture remained clear at pH 2.6, and it immediately became turbid after the pH of the solution was adjusted to pH 7.0 (Figure S6a,b). The generated aggregates appeared to contain nanofibrous structures as revealed by SEM visualization (Figure S6b). When the pH of the turbid CMP1-La3+ solution was readapted from 7.0 to 2.6, the aggregates got dissolved and the solution turned clear (Figure S6c,d). This suggested that the peptide CMP1-Ln3+ assembly was reversibly, finely regulated by pH.

Photoluminescence of the CMP1-Lanthanide Nanomaterials

Lanthanide ions may endow the peptide assemblies with extraordinary luminescent properties. Solid-state fluorescence was applied to characterize the photoluminescent features of the CMP1-Ln3+ assembled nanofibers (Figure ). The fluorescence emission spectrum of the CMP1-Eu3+ nanomaterials was obtained at an excitation wavelength of 394 nm and displayed characteristic peaks corresponding to the intra-4f65D0–7F0–2 transitions (Figure a). The observation of a strong line for 5D0–7F2 indicated that the CMP1-Eu3+ nanomaterials possessed good color purity, and the single line for 5D0–7F0 suggested the uniform chemical environment for Eu3+. The CMP1-Eu3+ assemblies were red-light emitters under UV irradiation. Photoluminescent features of the CMP1-Tb3+, CMP1-Dy3+, and CMP1-Gd3+ assembled materials were also characterized, and they behaved as pure green-light, yellow-light, and blue-light emitters, respectively (Figure b–d). Lanthanide ions have been reported to emit across the whole visible spectrum with high color purity.[50] Our results indicated that the peptide-Ln3+ assembled nanofibers exhibit attractive luminescent features, and their colors could be easily tuned by employing different types of lanthanide ions.

Figure 4

Fluorescence emission spectra of luminescent materials of peptide CMP1 with various lanthanide ions: Eu3+ (a), Tb3+ (b), Dy3+ (c), and Gd3+ (d). The emission spectra were measured at an excitation wavelength of 394, 370, 365, and 303 nm for Eu3+, Tb3+, Dy3+, and Gd3+, respectively. The inset shows photographs of the peptide CMP1-Ln3+ materials on a glass slide under a fluorescence microscope.

Morphology of Peptide CMP2-Lanthanide Nanomaterials

The morphology of the assembled nanomaterials of peptide CMP2 with lanthanide ion La3+ was evaluated by scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) (Figure ). The SEM image of the peptide CMP2-La3+ assemblies prepared in 50 mM HAc–NaAc buffer, pH 5.0, showed cross-linked nanofibers (Figure a,b). TEM visualization of the CMP2-La3+ assemblies revealed an interconnected three-dimensional fibrous framework (Figure c). Furthermore, the CMP2-La3+ assemblies showed the same regularly spaced bands as the CMP1-La3+ assemblies, demonstrating that the CMP2-La3+ nanomaterials recapitulated the unique banding pattern of native collagen (Figure d). The consistency of the distance between the banding gaps (∼10 nm) and the calculated length of the triple helix formed by peptide CMP2, reconfirmed the hypothesis of lanthanide-triggered linear as well as radial assembly of the peptide (Figure b).

Figure 5

Morphology of self-assembled peptide CMP2-La3+ nanomaterials. SEM images (a, b) and TEM images (c, d) of peptide CMP2 with lanthanide ion La3+. Peptide CMP2-La3+ nanomaterials were prepared in 50 mM HAc–NaAc buffer, pH 5.0.

The pH-responsive features of the CMP2-La3+ system were then evaluated under different pH conditions (Figure S7). Similar to peptide CMP1, the CMP2-La3+ mixture formed white aggregates in the range of pH from 4.0 to 7.0, while no visible aggregates were observed at pH 3.0 or lower. SEM images of the CMP2-La3+ aggregates obtained at pH 6.0 and 7.0 showed fibrous nanostructures, while their SEM images produced at pH 4.0 displayed short, brick-like nanostructures (Figure S7). These results demonstrated that pH mediated the capability of the peptide CMP2-La3+ assembly as well as the morphology of the final CMP2-La3+-assembled materials.

Biofunction of Self-assembled Peptide CMP2-Lanthanide Nanomaterials

The GFOGER motif within the triple-helical domain of collagen has been reported to recognize integrin receptors and mediate cell adhesion.[12] The biofunction of peptide CMP2-La3+ assemblies was thus evaluated by the cell adhesion assay using HeLa cells (Figure ). HeLa cells were cultured on the plate wells coated with heat-denatured bovine serum albumin (BSA), collagen, and CMP2-La3+ nanomaterials produced at four different pH conditions (pH 4.0, 5.0, 6.0, and 7.0), respectively. Cell adhesion to the native collagen substrate was taken as the 100% reference level. The percentage of the cell adhesion to BSA was 23%, indicating that HeLa cells barely bind to BSA substrates. The percentages of the cell adhesion to CMP1-La3+ nanomaterials produced at pH 4.0, pH 5.0, pH 6.0, and pH 7.0 were approximately 23%, 24%, 24% and 23%, respectively, demonstrating that HeLa cells barely bind to CMP1-La3+ substrates. In contrast, the percentages of the cell adhesion to CMP2-La3+ nanomaterials produced at pH 4.0, pH 5.0, pH 6.0, and pH 7.0 were approximately 62%, 62%, 62% and 63%, respectively, demonstrating that HeLa cells could efficiently attach onto the surfaces of CMP2-La3+ nanomaterials. Though the morphologies of CMP2-La3+ nanomaterials varied at different pH conditions, their cell adhesion activities were almost the same, indicating that the bioactive GFOGER motif played a determinant role for the CMP2-La3+ nanomaterials to recognize and interact with the receptors on the cell membrane surface.

Figure 6

Adhesion of HeLa cells as a function of surface composition: heat-denatured BSA, calf-skin collagen, CMP1-1 (CMP1-La3+ nanomaterials produced at pH 4.0), CMP1-2 (CMP1-La3+ nanomaterials produced at pH 5.0), CMP1-3 (CMP1-La3+ nanomaterials produced at pH 6.0), CMP1-4 (CMP1-La3+ nanomaterials produced at pH 7.0), CMP2-1 (CMP2-La3+ nanomaterials produced at pH 4.0), CMP2-2 (CMP2-La3+ nanomaterials produced at pH 5.0), CMP2-3 (CMP2-La3+ nanomaterials produced at pH 6.0), CMP2-4 (CMP2-La3+ nanomaterials produced at pH 7.0), CMP3-1 (CMP3-La3+ nanomaterials produced at pH 5.0), CMP3-2 (CMP3-La3+ nanomaterials produced at pH 6.0), and CMP3-3 (CMP3-La3+ nanomaterials produced at pH 7.0). Cell adhesion to collagen was used as a 100% reference level.

Cell adhesion and spreading properties were further examined by confocal fluorescence microscopy (Figures S8 and 7). The attached HeLa cells were fixed and stained for actin stress fibers and nuclei with phalloidin–tetramethylrhodamine isothiocyanate and Hoechst 33258 nuclear dye, respectively. The confocal images indicated that the HeLa cells on the CMP1-La3+-coated substrates produced at pH 4.0, 5.0, 6.0, and 7.0 maintained the spherical shape, and the cytoskeletal structure was poorly developed (Figure S8). In contrast, the confocal images indicated that the HeLa cells on the CMP2-La3+-coated substrates produced at pH 5.0 displayed a well-developed actin cytoskeletal structure (Figure a). The extensive cell adhesion and spreading demonstrated that the CMP2-La3+ nanofibers well mimic the cell interaction capability of natural collagen. In addition, the actin cytoskeletal organization remained the same in the cells seeded on CMP2-La3+ nanomaterials produced at pH 4.0, 6.0, and 7.0, suggesting that the morphologies of CMP2-La3+ nanomaterials did not affect the cell adhesion and spreading properties (Figure b–d). The extensive cell spreading was likely mediated by the integrin-cell adhesion process controlled by the GFOGER motif. These results suggested that the CMP2-La3+ nanofibers could assist cell adhesion and spreading.

Figure 7

Cell adhesion and spreading of HeLa cells as a function of the CMP2-La3+ nanomaterials produced at different pH conditions: pH 5.0 (a), pH 4.0 (b), pH 6.0 (c), and pH 7.0 (d). Cells were fixed and stained for actin stress fibers (red) and nuclei (blue). The images were recorded by confocal fluorescence microscopy.

Morphology and Biofunction of Self-assembled Peptide CMP3-Lanthanide Nanomaterials

The morphology of the assembled nanomaterials of peptide CMP3 with lanthanide ion La3+ was investigated by scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) (Figure ). The SEM image of the peptide CMP3-La3+ assemblies prepared in 100 mM Hepes buffer, pH 7.0, showed fibrous nanostructures (Figure a). TEM visualization of the CMP3-La3+ assemblies revealed the characteristic periodic bands of ∼10 nm (Figure b). These results demonstrated that peptide CMP3 shared the same capability as CMP1 and CMP2 to facilitate the head-to-tail as well as radial assembly triggered by lanthanide ions (Figure b). The pH-responsive features of the CMP3-La3+ system were then examined under different pH conditions (Figure S9). The CMP3-La3+ mixture formed white aggregates in the range of pH from 5.0 to 7.0, while no visible aggregates were observed at or below pH 4.0. SEM images of the CMP3-La3+ aggregates obtained at pH 5.0 and 6.0 displayed much shorter segments (Figure S9). These results indicated that the peptide CMP3-La3+ assembly was modulated by pH.

Figure 8

Morphology and function of self-assembled peptide CMP3-La3+ nanomaterials. The SEM image (a) and TEM image (b) of peptide CMP3-La3+ nanomaterials prepared in 100 mM Hepes buffer, pH 7.0. Cell adhesion and spreading of HeLa cells of the CMP3-La3+ substrates (c,d). Cells were fixed and stained for actin stress fibers (red) and nuclei (blue).

The biofunction of peptide CMP3-La3+ assemblies was examined by the cell adhesion assay (Figure ). The percentages of the cell adhesion to CMP3-La3+ nanomaterials produced at pH 5.0, pH 6.0, and pH 7.0 were approximately 66%, 67% and 71%, respectively, demonstrating that HeLa cells could efficiently attach onto the surfaces of CMP3-La3+ nanomaterials (Figure ). Cell adhesion and spreading features of CMP3-La3+ nanomaterials were further characterized by confocal fluorescence microscopy (Figure c,d). The confocal images indicated that the HeLa cells on the CMP3-La3+-coated substrates produced at pH 7.0 displayed a well-pronounced actin cytoskeletal structure (Figure c,d). Similar cell adhesion performance was observed for the CMP3-La3+-coated substrates produced at pH 5.0 and 6.0 (Figure S10). The extensive cell adhesion and spreading demonstrated that the CMP3-La3+ nanofibers well recapitulate the cell interaction capability of native collagen. These results indicated that both the CMP2-La3+ and CMP3-La3+ nanofibers facilitate cell adhesion and spreading.

Conclusions

Collagen has been a hot target for biomimetic design due to its excellent structural and functional properties.[21] A number of triple-helical peptide-based strategies such as electrostatic and hydrophobic interactions have been established to generate collagen mimetic nanofibers.[20] Though significant advances have been achieved to construct self-assembling peptides to recapitulate the nanofibrous structure of native collagen, there are far fewer reports to fabricate peptide assemblies that possess both structural and biofunctional features of collagen.

We have recently discovered that a collagen-like peptide DD(PPG)12DD can be initiated by lanthanide ions to form nanoropes.[22] Herein, by introducing a central Asp in the peptide CMP1 as an additional binding unit of lanthanide ions to trigger radial assembly besides the head-to-tail assembly assisted by the terminal aspartic acids, we have created exquisite fibrous nanomaterials with distinct periodic banding patterns, which well mimic the hierarchical structure of natural collagen. It agrees well with previous studies that the morphology of divalent metal-triggered peptide assemblies depends on the numbers and locations of metal-binding units.[25,29,36] The successful Ln3+-triggered assembly of peptides, CMP2 and CMP3, demonstrates that the simultaneous incorporation of negatively charged amino acids in the middle as well as at N- and C-terminals of triple-helical peptides provides a reliable and robust strategy to create collagen mimetic nanofibers. Compared with glutamic acids, aspartic acids have shorter side chains, which may result in better coordination with lanthanide ions, leading to good fibrous nanostructures.

The assembly of all of the three constructed collagen mimetic peptides has been shown to be specifically mediated by lanthanide ions. The utilization of lanthanide ions provides a convenient external stimulus to ensure that the peptide self-assembly is an easily controlled and well programmable process, and it also functionalizes collagen mimetic materials with easily tunable photoluminescence. Due to the excellent features such as line-like emission, long luminescence lifetime, and high photochemical stability, lanthanide ions have been widely applied as photoluminescent materials in cell imaging and medical diagnostics.[50] The handy selection of different kinds of lanthanide ions will greatly enhance the luminescent properties of the hybrid peptide-Ln3+ nanomaterials.

Interestingly, this constructed peptide-Ln3+ system well mimics the pH-dependent fibrillogenesis of Type I collagen.[49,51] The aggregation tendency and the morphology of final assembled materials for the peptides and type I collagen are modulated in a similar pH-cooperative manner. The sensitivity to environmental pH has been considered to play an essential role in the collagen structure and function.[49] This peptide-Ln3+ system provides a facile approach to create peptide-based nanofibers that well recapitulate pH-responsive features of native collagen. It probably results from the pH-dependent binding behavior of negatively charged amino acids and lanthanide ions. At a neutral pH, the deprotonated aspartic acids or glutamic acids facilitate strong binding of Asp/Glu-Ln3+, leading to the generation of self-assembled nanomaterials. However, at too acidic pH, the protonated amino acids become poor ligand for Ln3+, which is unable to initiate the self-assembly of the peptides.

Most notably, the lanthanide-triggered peptide assembled nanomaterials possess good cell adhesion properties characteristic of collagen. The inclusion of integrin-binding motif GFOGER in triple-helical peptides CMP2 and CMP3 enables these peptide assemblies to recognize and bind with HeLa cells as shown by the cell adhesion assay. The presence of charged amino acids Glu and Arg appears not to interfere in the appropriate assembly of the peptides. The confocal images show extensive cell adhesion and spreading on the surface of these peptide-La3+ substrates, demonstrating that the peptide-La3+ nanofibers well mimic the cell interaction properties of native collagen. To conclude, triple-helical peptides supplemented with the bioactive GFOGER motif and the simultaneous incorporation of negatively charged amino acids in the middle as well as at the terminals provide a convenient and potent approach to generate nanofibers that mimic both structural and functional features of native collagen. These novel pH-responsive, luminescent, and biofunctional collagen mimetic nanofibers open fascinating opportunities in the development of improved functional biomaterials in tissue engineering, drug delivery, and medical diagnostics.

Experimental Section

Peptide Synthesis

Peptides CMP1 and CMP2 were obtained from the Chinese Peptide Company (Hangzhou, China). Peptide CMP3 was synthesized following the Fmoc solid-phase synthesis method at a 0.1 mmol scale. 2-Chlorotrityl chloride resin was used. Stepwise coupling of amino acids was carried out according to a double coupling protocol using diisopropylethylamme (6 eqiuv), Fmoc-amino acids (4 eqiuv), and activator reagents (HBTU + HOBt 0.66 mmol/ml, 4 eqiuv). Dichloromethane (3 × 5 mL2) and dimethylformamide (DMF) (3 × 5 mL2) were employed to wash the reaction mixture after each step of coupling, and 20% piperidine was added to remove the Fmoc protection group. A test reagent (2% chloranil DMF, 2% ethanal DMF) was applied to monitor the process of Fmoc deprotection and coupling reaction. The resin was treated with TFA/TIS/H2O (95:2.5:2.5) for 3 h to eliminate the tBu groups and to take the peptides off the resin. The peptides were precipitated by Cold Et2O, collected, and resuspended in Et2O. After sonication and centrifugation, crude products were harvested, dissolved in water, and lyophilized. The peptides were purified by reverse-phase high-performance liquid chromatography, and their identity was determined by mass spectrometry. m/z calculated 3511.8 [M]+ for peptide CMP1, found 3510.9 [M]+; m/z calculated 3846.9 [M]+ for peptide CMP2, found 3846.0 [M]+; m/z calculated 3931.3 [M]+ for peptide CMP3, found 3931.3 [M]+.

Sample Preparation

Fresh solutions of CMP1 with a starting concentration of 5.0 mg/mL were prepared under different pH conditions: pH 7.0 (100 mM Hepes buffer), pH 6.0–6.4 (100 mM 2-(N-morpholino)ethanesulfonic acid buffer), and pH 2.6–5.0 (100 mM HAc–NaAc buffer). Fresh solutions of CMP2 and CMP3 with a starting concentration of 5.0 mg/mL were prepared under various pH conditions: pH 7.0 (100 mM Hepes buffer) and pH 3.0–6.0 (50 mM HAc–NaAc buffer).

Circular Dichroism Spectroscopy

Circular dichroism (CD) spectra were measured on an Aviv model 400 spectrophotometer (Applied Photophysics Ltd, England) equipped with a Peltier temperature controller. The CMP1 samples (142 μM) were prepared in 20 mM Tris-HCl buffer at pH 7.0. Wavelength scans were recorded from 210 to 260 nm at 4 °C. The CMP2 and CMP3 samples (300 μM) were prepared in 20 mM phosphate buffer at pH 7.0, and wavelength scans were performed from 210 to 280 nm. The increment per step was 0.5 nm and averaging time was 0.5 s. Each measurement was performed three times.

The thermal stability of the peptides was determined by measuring the intensity of the CD peak at 225 nm when the temperature was gradually increased from 4 to 85 °C. The average heating rate was 0.4 or 0.5 °C/min and the equilibration time was 2 min. The peptides were heated at 90 °C for 20 min and incubated at 4 °C for >24 h. The first derivative of the unfolding curve was computed and smoothed by the Savitzky–Golay method. The melting temperature (Tm) was estimated as the extrema of the first derivative.

Scanning Electron Microscopy

SEM images of the peptide-Ln3+ nanomaterials were recorded on a Hitachi S-4800 scanning electron microscope (Hitachi Limited, Japan). The operating voltage was 5.1–5.3 kV. The peptide CMP1 solution (1.42 mM, 100 mM Hepes buffer, pH 7.0) was incubated with lanthanide ions at different molar ratios of peptide/Ln3+ in the range of 1:1 to 1:5 at 25 °C for 48 h. The peptide solutions of CMP2 and CMP3 (1.3 mM, 100 mM Hepes buffer, pH 7.0) were incubated with lanthanide ions at 25 °C for 24 h, respectively. The peptide-Ln3+ mixtures with observable white precipitates were centrifuged. The precipitates were collected, washed with ultrapure water, and resuspended in ethanol. 20 μL of the resuspended solution was dried on a silicon slice and then sputter-coated with AuPd for 20 s before imaging.

Transmission Electron Microscopy

TEM images were obtained using a JEM-2100 transmission electron microscope (JEOL company). The operating voltage was 200 kV. The precipitates of the peptide-Ln3+ mixture were prepared by the same steps as the SEM measurements. The resuspended solution (10 μL) was deposited on a copper grid coated with the carbon film and kept for three minutes to allow deposition. After carefully removing excessive buffer by filter paper, the copper grid was negatively stained using 2.0% uranium acetate or 2.5% phosphotungstic acid. The grid was dried for >12 h under ambient conditions.

Fluorescence Spectroscopy

Fluorescence spectra were recorded on a Hitachi FLS920 spectrofluorometer (Edinburgh Instruments company). A monochromated Xe lamp was used as an excitation source. The peptide CMP1-Ln3+ (Eu3+, Tb3+, Dy3+, and Gd3+) aggregates were collected after incubation at 25 °C for 48 h. The emission spectra were measured for the peptide-Ln3+ (Eu3+, Tb3+, Dy3+, and Gd3+) nanomaterials, and the excitation wavelengths were 394, 370, 365, and 303 nm, respectively. Photographs were recorded with a DMI4000B LEICA inverted fluorescence microscope.

Cell Adhesion Assay

Ninety-six-well microtiter plates (non-tissue culture) were coated with native Type I collagen, peptide CMP1-La3+, CMP2-La3+, and peptide CMP3-La3+ nanomaterials, respectively. The control wells of the plate were coated with heat-denatured BSA. Phosphate buffered saline buffer (PBS) (10 mM) was then used to wash the wells three times before culturing the Hela cells. 100 μL of cell suspension in serum-free Dulbecco’s modified Eagle’s medium (1 × 106 cells/mL) was added into the wells and grown at 37 °C for 4 h. Unattached cells were removed by washing using PBS buffer (10 mM).

The amount of attached cells were determined by a total deoxyribonucleic acid (DNA) quantification assay (Hoechst 33258, Solarbio). Briefly, the adhered cells in each well were lysed by three freeze-thaw cycles using ultrapure water. Hoechst 33258 with a final concentration of 5 μg/mL was applied to the cell lysates and kept in the dark for 1 h. A microplate reader (Tecan Infinite M200) was employed to measure the fluorescence, while the excitation and emission wavelengths were 360 and 465 nm, respectively. The assays were carried out in triplicate.

Immunofluorescence Staining

Fluorescent confocal dishes (Nontreated) were treated with one thin layer of CMP1-La3+, CMP2-La3+, and CMP3-La3+ substrates, respectively. PBS buffer (10 mM, pH 7.2–7.4) was used to wash the coverslips 3 times before culturing the cells. HeLa cells with a density of ∼600 cells/mm2 in Dulbecco’s modified Eagle’s medium were added on the coverslips and nurtured at 37 °C for 4 h. Cold 4.0% formaldehyde and 0.1% Triton X-100 were used to fix and permeabilize the attached cells for 10 min and 5 min, respectively. 1% BSA in PBS buffer (10 mM, pH 7.2–7.4) was then applied as a blocking agent for 0.5 h at room temperature. The cells were mixed with 1 mL of 100 nM phalloidin–tetramethylrhodamine isothiocyanate (Solarbio, China) (in 10 mM PBS, pH 7.2–7.4)) for 1 h. The cell actin cytoskeleton and cell nucleus were then stained by the addition of 1 mL 5 μg/mL Hoechst 33258 (Sigma-Aldrich) (in ultrapure water). The images were recorded on OLYMPUS Fluoview FV3000.