Article pubs.acs.org/journal/abseba
Thermally Controlled Collagen Peptide Cages for Biopolymer Delivery Jeremy Gleaton and Jean Chmielewski* Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907, United States S Supporting Information *
ABSTRACT: The hierarchical assembly of collagen-based triple helical peptides into disks, followed by metal-promoted assembly into microcages is described. The length of the triple helix was found to correlate to the height of the disks that formed, providing mechanistic insights into their formation. The encapsulation of fluorescently labeled dextrans within the peptide microcages, and their subsequent thermal release is detailed. The half-life for thermal release of the encapsulated cargo from the cages was found to increase as the peptide triple helix stability increased, providing a means to engineer cargo delivery rates through peptide design. KEYWORDS: collagen peptide, microcage, hierarchical assembly, encapsulation, thermal release
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also elicits.5 More recently platelet aggregation was also demonstrated with a collagen peptide hydrogel that was achieved through salt bridge interactions.22 Moreover, the hydrogel displayed minimal hemolysis and was noninflammatory. Effective encapsulation and growth of cells within a threedimensional scaffold generated by metal promoted assembly of collagen peptide triple helices has also been described.20,21 The modular nature of this assembly allowed for the incorporation of biological cues, such as cell adhesion signals to promote tissue growth,21 whereas microflorettes bind and release Histagged proteins.23 These examples highlight the usefulness of materials based on collagen peptides and demonstrate that the structures can be implemented as alternatives to materials fabricated from natural collagen. The collagen peptide Hbyp3 (Figure 1A) was of particular interest to us for use as a biofunctional material. This peptide has been shown to undergo heirarchical assembly of its triple helix into nano- to microscale disks. The addition of metal ions, such as Fe(II) allowed the disks to assemble into small micronscaled hollow spheres.19 Herein we detail the design of an extended collagen based peptide with an additional Pro-HypGly unit at each temini, Hbyp3−11 (Figure 1A), to probe the mechanism of disk assembly and the subsequent formation of hollow spheres from the disks in the presence of metal ions. The use of the cages formed with both of these collagen peptides for the encapsulation and thermal release of biopolymers is detailed within.
INTRODUCTION Collagen serves a significant role in tissue development and regeneration in mammals. As such, recapitulation of the structure and function of collagen is a high priority for areas such as regenerative medicine and drug delivery. One method to accomplish this task is the use of self-assembling collagenbased peptides to mimic natural collagen.1−3 A range of strategies has been developed to promote higher order assembly of collagen peptide triple helices, including hydrophobic interactions,4 cation-π interactions,5 cysteine knots,6−8 native chemical ligation,9 electrostatic interaction,10,11 and metal ligand interactions.12 Many of these collagen peptides assemble into fibrillar structures, whereas other cues such as encoded electrostatic interactions13,14 and stereochemistry and shape complementarity15 also permit the formation of sheets. Depending on the placement of ligands for metal ions within collagen peptide triple helices, a number of other nonfibrillar structures have also been obtained. For instance, ligands at the termini of the triple helix generated linear assemblies in the presence of metal ions, resulting in microflorettes16 and petallike structures with nanoscale banding.17 Placement of ligands at the center of the triple helix resulted in nano- to microscale disks with18 and without the addition of metal ions, the latter providing hierarchical assembly into hollow spheres when metal ions were then introduced.19 Co-placement of ligands at both the center and termini of collagen peptides resulted in a highly cross-linked morphology that is very similar to the structure of Matrigel, a commonly used material that mimics the threedimensional matrix.20,21 In a few cases, the assembled structures that resulted from collagen peptide triple helices have been evaluated as biofunctional materials. Assembly of collagen triple helices via aromatic interactions provided micrometer sized fibers, that induced platelet aggregation, a function that natural collagen © XXXX American Chemical Society
Received: June 5, 2015 Accepted: September 1, 2015
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DOI: 10.1021/acsbiomaterials.5b00241 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
Figure 1. (A) Molecular representations of Hbyp3 and Hbyp3−11. (B) Circular dichroism spectra indicating the maxima at 225 nm for both Hbyp3 and Hbyp3−11 (150 μM, 10 mM HEPES, pH 7.0). (C) First derivative of melting curves to determine the Tm values for Hbyp3 (42 °C) and Hbyp3−11 (62 °C).
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RESULTS AND DISCUSSION The Hbyp3 and Hbyp3−11 peptides were synthesized using an Fmoc-based solid-phase peptide synthesis approach as previously described.19 Briefly, the designed peptides were synthesized on ChemMatrix Rink Amide resin with HBTU as the coupling reagent. A lysine residue with the acid labile 4methyltrityl (Mtt) protecting group at the ε-position was incorporated at the appropriate points within the sequences. Upon completion of the synthesis of the full length peptides, the Mtt groups were removed on resin with 1.8% trifluoroacetic acid (TFA) in dichloromethane (DCM) and the resulting amino groups were functionalized with 4′-methyl-2,2′-bipyridine-4-carboxylic acid24 using HBTU. The peptides were cleaved from the resin using 95% TFA, 2.5% water, and 2.5% triisopropylsilane. The resulting material was purified to homogeneity using reverse-phase HPLC and characterized by MALDI-TOF mass spectrometry. We hypothesized that Hbyp3−11 would form a more stable triple helix than Hbyp3 because the former has two additional Pro-Hyp-Gly units, a feature that has led to increased stability in a number of collagen mimetic peptides.25 Circular dichroism (CD) was used to determine if Hbyp3−11 adopted a collagen triple helix and to ascertain its thermal stability. A maximum at 225 nm was observed for solutions of Hbyp3−11 (Figure 1B), a hallmark of the polyproline type II helix that makes up a collagen triple helix. When this wavelength was monitored during a temperature sweep from 4 to 90 °C a cooperative
dissociation was observed as has been observed for other collagen triple helical peptides,26 providing a melting temperature (Tm) of 62 °C for Hbyp3−11 (Figure 1C). These data support the formation of collagen triple helix with increased stability as compared to Hbyp3 (Tm of 42 °C). The self-assembly of Hbyp3−11 without added metal ions was monitored using dynamic light scattering (DLS) and compared to Hbyp3. Experiments were performed with solutions of peptide (250 μM, 10 mM HEPES buffer, pH 7.0) that were thermally annealed by heating to 90 °C for 30 min, followed by incubation at 4 °C for 48 h. Under these conditions both peptides were found to refold into a triple helix by CD (see Figure S2). Assemblies with diameters of approximately 1 μm were noted for both Hbyp3 and Hbyp3−11 in the absence of metal ions (Figure 2). We hypothesized that these larger aggregates form via aromatic interactions between the bipyridyl units of individual collagen triple helices. To test this hypothesis, the annealing experiments were performed using an acidic buffer (10 mM glycine buffer, pH 3.0) to protonate the bipyridine ligands and potentially impede the aromatic interactions. DLS analysis demonstrated significantly smaller aggregates (Figure 2), with both peptides having diameters that correspond to values observed for individual collagen triple helices.19,27 The increase in the particle sizes observed by DLS for Hbyp3−11 under acidic conditions, as compared to Hbyp3, can be attributed to the increase in peptide length from the additional two Pro-Hyp-Gly B
DOI: 10.1021/acsbiomaterials.5b00241 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
Atomic force microscopy (AFM) was used to image the aggregates formed with Hbyp3−11. Samples of the thermally annealed peptide displayed a circular morphology that is very similar to that observed with Hbyp3 (Figure 3A and B). A cross-section analysis of these structures showed them to be disks with heights of 13 ± 0.5 nm and 11 ± 0.4 nm for Hbyp3−11 and Hbyp3, respectively (Figure 3C, averages determined from cross section analysis of 15 disks). This average difference of ∼2 nm in the disk height is very close to the expected increase in the triple helix length with the addition of two Pro-Hyp-Gly units at the termini of Hbyp3−11.28,29 These data support a structure for the disks in which the triple helices of the collagen-based peptides are standing upright on the surface of the AFM grid, as has been observed for nanosheets composed of collagen peptide triple helices.13 In our model for the formation of the disk structures, bipyridine ligands should be displayed along the side edges of the disks. Therefore, metal-promoted assembly of the Hbyp3− 11 disks (250 μM) was investigated with FeClO4 (250 μM). The peptide-metal ion solution was incubated for 48 h, and the pink precipitate that formed was collected, washed, and imaged using transmission electron microscopy (TEM). Hbyp3−11 formed structures that resembled collapsed cages ranging in size from 2 to 5 μm (Figure 4A and B) as was previously
Figure 2. (A) Dynamic light scattering (DLS) data for Hbyp3 and Hbyp3−11, 10 mM HEPES, pH 7.0 (red), and 10 mM glycine, pH 3.0 (blue). (B) Table summarizing the size aggregates for Hbyp3 and Hbyp3−11 (250 μM) as determined by DLS.
Figure 4. Representative TEM micrographs of the assemblies formed upon addition of Fe(ClO4)2 (250 μM) to (A) Hbyp3 and (B) Hbyp3−11 (250 μM peptide in 10 mM HEPES buffer, pH 7.0, stained with 4% uranyl acetate) (scale bars 2 μm).
units. The Tm values obtained for the peptides by CD, therefore, correspond to melting of triple helices within the assembled disks. CD experiments at pH 3 (conditions that favor isolated triple helices), however, provide very similar melting temperatures of 58 and 43 °C for Hbyp3−11 and Hbyp3, respectively, (see Figure S3), indicating that assembly does not significantly change the melting behavior of the collagen peptide triple helices.
observed with Hbyp3,19 confirming that the additional ProHyp-Gly units did not interfere with the metal-promoted assembly. We have also investigated substoichiometric levels of Fe(II) in the assembly process (0.1 and 0.5 equiv with respect to the peptides). Fully formed cages were observed under both
Figure 3. Atomic force microscopy images of the collagen peptides after thermal annealing in 10 mM HEPES buffer, pH 7.0. (A) Hbyp3 (250 μm) and (B) Hbyp3−11 (250 μm) (scale bars 2 μm). (C) Representative graphs comparing the height differences for Hbyp3 and Hbyp3−11 from the AFM data in A and B. C
DOI: 10.1021/acsbiomaterials.5b00241 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
could be degraded and the contents released by thermally melting the triple helices. On the basis of the melting temperatures of the peptides, we hypothesized that the cages formed with Hbyp3 would thermally degrade and release cargo more readily as compared to Hbyp3−11, since the latter has a higher Tm value. Release of the 40K and 3K dextrans from the hollow spheres was monitored over a 24 h period at 37 and 60 °C (Figure 6). These temperatures were chosen to provide differing levels of folding of the Hbyp3 and Hbyp3−11 triple helices (about 65 and 90% folded at 37 °C, and about 20 and 50% folded at 60 °C, respectively). At 37 °C, no 40K dextran release was observed from either of the cages formed from Hbyp3 and Hbyp3−11 after a 24 h time period, and only low levels of release were observed (
