Self-Assembled Water-Soluble Nanofibers Displaying Collagen

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Self-assembled water-soluble nanofibers displaying collagen hybridizing peptides Boi Hoa San, Jeongmin Hwang, Sujatha Sampath, Yang Li, Lucas L. Bennink, and S. Michael Yu J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b07900 • Publication Date (Web): 01 Nov 2017 Downloaded from http://pubs.acs.org on November 1, 2017

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Self-assembled water-soluble nanofibers displaying collagen hybridizing peptides

Boi Hoa San1, Jeongmin Hwang1, Sujatha Sampath1, Yang Li1 Lucas L. Bennink1 and S. Michael Yu1,2 1. Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA

2. Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112, USA Email: [email protected]

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Abstract Collagen hybridizing peptides (CHP) have been demonstrated as a powerful vehicle for targeting denatured collagen (dColl) produced by disease or injury. Conjugation of β-sheet peptide motif to the CHP results in self-assembly of nonaggregating β-sheet nanofibers with precise structure. Due to the molecular architecture of the nanofibers which puts high density of hydrophilic CHPs on the nanofiber surface at fixed distance, the nanofibers exhibit high water solubility, without any signs of intramolecular triple helix formation or fiber-fiber aggregation. Other molecules that are flanked with the triple helical forming GlyProHyp repeats can readily bind to the nanofibers by triple helical folding, allowing facile display of bioactive molecules at high density. In addition, the multi-valency of CHPs allows the nanofibers to bind to dColl in vitro and in vivo with extraordinary affinity, particularly without pre-activation that unravels the CHP homotrimers. The length of the nanofibers can be tuned from micrometers down to 100 nm by simple heat treatment, and when injected intravenously into mouse, the small nanofibers can specifically target dColl in the skeletal tissues with little target-associated signals in the skin and other organs. The CHP nanofibers can be a useful tool for detecting and capturing dColl, understanding how ECM remodelling impacts disease progression, and development of new delivery systems that target such diseases.

Keywords: Collagen hybridizing peptide, self-assembly, nanofibers, β-sheet, drug delivery, denatured collagen.


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Introduction As the most abundant protein in mammals, collagen is the fundamental component of the extracellular matrix (ECM), supporting cell proliferation, differentiation, and regeneration.1 Since abnormality in collagen metabolism can signify pathologic conditions, collagen has been explored as a target for diagnostic imaging and therapeutic delivery. So far, many efforts have been made to the design and improvement of probes for targeting the native collagen in the ECM.2-8 For instance, small peptides selected from phage display or derived from small domains of collagen binding proteins have been exploited for imaging,2 drug delivery,3-5 and tissue engineering.6-8 Unfortunately, most of these peptides that target fibrous collagens with high triple helical content suffer from low affinity and nonspecific binding, because the tightly folded triple helical structure of collagen is not conducive to molecular recognition and strong binding.2,9 On the other hand, our research group has demonstrated that the unfolded single strand of collagen can be selectively targeted by collagen hybridizing peptides (CHPs) in a manner similar to a primer binding to a melted DNA strand during PCR. Since partially degraded and denatured collagens (dColl) are found in many pathologic conditions, such as arthritis, osteoporosis, cancer and other chronic wounds,10,11 the ability to seek and bind unfolded collagen strands may help us reveal new disease mechanisms or develop new theranostics for managing such diseases. CHPs are a family of synthetic peptides that have a strong propensity to fold into a collagen triple helix. Previously, we discovered that the CHP of sequence (GPO)n (n = 6-10, G = Glycine, P = Proline, O = hydroxyproline) binds to dColl by hybridizing with single collagen strands and re-forming the triple helical structure.12,13 CHPs are



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comprised of neutral and hydrophilic G, P, O residues which are biochemically inert,14 but have a strong propensity to assemble into a triple helix. When CHPs are presented in their monomeric form, they exhibit high selective affinity to dColl strands having triple helical G-X-Y sequence. Since CHPs in the homo-trimeric triple helical form are not able to interact with collagen strands, the ability to curb self-trimerization while maintaining triple helical propensity is crucial for the practical use of CHPs in biomedical applications.10,13,15-17 Currently, there is no pure peptide-based CHP system with non-trimerizing CHPs that are capable of hybridizing to dColl strands, although a few strategies have been developed to circumvent CHP self-trimerization for practical use.10,13,18,19 Because of slow cis-trans isomerization speed of proline, (GPO)9 self-trimerizes at a very slow rate with a half-life on the order of tens of min at µM concentrations.20,21 Therefore, a CHP which is heated above its Tm can be cooled quickly to room temperature or body temperature and used immediately without losing its ability to hybridize to dColl strands. Although this is a simple strategy, it is nearly impossible to determine the exact concentration of active CHP strands that are used in such experiments, which poses a significant hurdle in translating the technology for human use. In another strategy, a photo-cleavable nitrobenzyl (NB) group was conjugated to the central glycine in the (GPO)n sequence which effectively prevented the triple helical folding via steric hindrance.13,22,23 This peptide, also known as caged-CHP, can be triggered to fold into a triple helix by UV irradiation which cleaves the NB group.13,22,23 Although this is an excellent way to trigger binding, the side product of NB cleavage can be toxic when used in vivo, and the uncertainty in the concentration of active monomeric CHP is still a


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problem, because some UV-activated CHP will self-trimerize during usage. Therefore, we had long-standing interests in designing a new generation of CHPs that can target dColl with high specificity and affinity, without the need for preconditioning process such as heat or UV irradiation.12,14,24,25 Our prior work on CHPs immobilized on nanoparticles (NPs) provided a key insight into the design of a new CHP that does not self-trimerize but can bind to collagen strands.25 When polymers with CHP side chains were adsorbed to the surface of nanoparticles, the CHPs did not self-trimerize because the adsorbed polymer backbone prevented the CHPs from interacting freely. The adsorption process seemed to have left the individual CHPs physically separated, not allowing homotrimer formation, but the CHPs were still able to hybridize with dColl strands. Although the NP system provided the first success in realizing the non self-trimerizing CHP system, it is a highly heterogeneous and complex system unsuitable for in vivo use, and its making involves multi-step process from conjugating CHP to polymer, to mixing with another type of conducting polymer and precipitation under specific condition. We reasoned that a supramolecular structure that displays multiple CHPs at a fixed distance could keep CHPs from self-trimerization while the multi-valency of the CHPs would significantly increase the collagen binding affinity. We thought that such system in its simplest form could be constructed from self-assembling peptides. Previously, collagen mimetic peptides (CMPs) have been utilized as building blocks for the design of self-assembled nanostructures such as nanofibers,26-29 nanosheets,30-33 dendrimers,34,35 and vesicles.36,37 However, those constructs were designed to mimic the natural nanostructure of the ECM molecules by using the triple helical folding as means for the assembly process, and their



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interactions with collagen, which is not likely given the pre-formed triple helix, have never been tested. In this study, we introduce for the first time a CHP-based self-assembled peptide nanofiber that can specifically target dColl with high efficiency. The nanofiber is comprised of a core of extended β-sheet structure, which is a well-known structural motif for nanostructure assembly. Because the β-sheet nanofiber is decorated with a dense layer of CHPs that are in a fixed position with a defined inter-CHP distance, the CHPs remain single strand, allowing the fibers to readily bind to dColl both in vitro and in vivo with surprisingly high affinity, even without any pre-conditioning such as heat treatment or UV irradiation.

Here we report the peptide design, the molecular structure of the

nanofiber, and its remarkable affinity to dColls in vitro and in vivo which demonstrates their potential for applications in the management of diseases characterized by high collagen remodeling activity.

Results and discussion In order to transform CHPs into nanofibers, we attached CHP to the N-terminus of a short β-sheet forming peptide with a triple glycine (G3) spacer in a form of (GPO)9-G3(FKFE)2 [designated as NF-1]. (FKFE)2 is a well known β-sheet motif comprised of an alternating sequence of hydrophobic and hydrophilic amino acids. This peptide forms self-assembled hydrogel that consists of fibrillar networks of extensive bilayer β-sheet structures.38,39 Even when other short peptides or small molecules are conjugated, the (FKFE)2 is still able to form nanofiber structures, partly due to its strong propensity to form β-sheets.40,41 Therefore, we expected that NF-1 would assemble into fiber structure


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with the stacked β-sheet occupying the core of the fiber and the CHPs decorating the surface as shown in Figure 1. NF-1 was prepared by conventional automated solid phase peptide

synthesis

and

purified

by

reversed

phase

high-performance

liquid

chromatography (RP-HPLC). The purified peptide was lyophilized, and the powder was dissolved in water to make 2 mM solution which was clear with no visible sign of aggregation or gel formation. We first analyzed the secondary protein structure of NF-1 in aqueous solution by circular dichroism (CD) and fourier-transform infrared spectroscopy (FTIR) spectroscopy. The CD profile showed a typical β-sheet band at 215 nm and an additional negative adsorption at 202 nm (Figure 2A),42 which can be attributed to the aromatic π-π stacking interactions from the phenylalanine residues. CD melting experiments had no clear melting transition for a triple-helix, and instead showed a linear decrease in ellipticity at 225 nm, indicating a lack of thermal transition43 (Figure 2B). Even though NF-1 folds into β-sheet fiber structure, the CHPs on the fiber surface did not trimerize and likely remained as polyproline-II helix. Otherwise, we would have detected an inflection point at around 74 °C corresponding to the Tm of (GPO)9. Furthermore, the FTIR showed amide I absorption peaks at 1625 and 1695 cm-1 which are classic signatures of an antiparallel β-sheet structure (Supplementary Information, Figure S1). These results are in agreement with previously reported antiparallel β-sheet forming peptides.42,44 NF-1 self-assembles into antiparallel β-sheet nanostructures nearly identical to the (FKFE)2 nanofibers without the formation of triple helices. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) confirmed that NF-1 self-assembles into precisely-defined nanofiber structure (Figure 1)



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in aqueous solution (Figure 3). The micrographs showed the presence of unaggregated long nanofibers that were approximately 22 nm in width and 1.5 nm in height (Figure 3) which are comparable to the calculated dimension (20.4 nm × 1.1 nm) assuming antiparallel β-sheet stacks of the bilayer units and CHPs in polyproline-II helix conformation.45 The molecular structure of NF-1 was further studied by x-ray diffraction experiments (Figure 4). The x-ray diffraction showed two major peaks, a sharp intense peak at 4.6 Å spacing and a weak broad peak at 11.2 Å spacing, which correspond to the distance between two adjacent β-strands within the β-sheet and the distance between two β-sheets in the bilayer, respectively (Figure 1). These values are comparable to previously reported values for (FKFE)2 nanofibers obtained from a different measurement method.42 The inter-sheet spacing of 11.2 Å is close to the height of the nanofiber (15 Å) measured by the AFM (Figure 3), further corroborating the formation of the bilayer nanofibers. Compared to the x-ray diffraction of (FKFE)2 (NF-C, Figure S2), the 11.2 Å peak is broader because the CHP part of the peptide interfered with the stacking of the nanofibers creating more disorder. According to the molecular structure of the nanofiber, the closest distance between two neighboring CHPs is calculated to be 9.2 Å which is the distance between the CHPs in the same sheet separated by one antiparallel peptide. Since the inter-strand distance need to be less than 4.0 Å to form a stable triple helix, it is very likely that the CHPs on the nanofibers are in a single strand form, although the flexible triple Gly spacer may allow some level of partial overlap between the CHPs.45 Collectively, these findings confirm that the NF-1 nanofibers have antiparallel β-sheet


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bilayer structures, and the CHPs remain in their unfolded single strand state due to the spatial separation between CHPs. To our surprise, NF-1 exhibited remarkable water solubility which was unusual for a nanofiber based on a β-sheet structure. NF-1 dissolved in aqueous solutions without any sign of aggregation in a wide range of conditions. Conventional nanofibers derived from β-sheet peptides easily aggregate and turn into hydrogels, due to extensive fiberfiber interactions,26,46-51 unless the hydrophobicity of the beta-sheet core is carefully balanced with hydrophilic domains.44 NF-1 showed no significant aggregation in aqueous solutions over a wide pH range and concentrations up to 5 mM (Figure S3-A), as confirmed by CD and TEM (Figure S4 and S5). On the contrary, the parent peptide (FKFE)2, designated as NF-C, formed large aggregates and eventually turned into a hydrogel under similar conditions (Figure S6). CHPs can self-trimerize when they are in a parallel orientation but when in an antiparallel orientation, they have a tendency to repel each other. Therefore, when two NF-1s approach each other side by side, they will repel due to the antiparallel orientation of CHPs from the two nanofibers (Figure 1). In addition, CHP is a hydrophilic and inert peptide comprised of only neutral hydrophilic amino acids. It is the hydrophilic CHPs passivating the NF-1 which conferred such high water solubility to the nanofibers. We did not observe any aggregation of NF-1 in 1× PBS (pH 7.4) at a concentration of 2 mM (Figure S5-B); however, we did notice aggregation of NF-1 in 1×PBS at 5 mM and above (Figure S3-B). Considering the fact that such aggregation was not observed in pure water (Figure S3-A), we believe that the increased hydrophobic interactions made possible by the high ion strength that reduces the Debye screening length caused the NF-1 to aggregate. This finding is also consistent



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with previously reported (FKFE)2 derived nanofibers and similar β-sheet forming peptide nanofibers..52-54 Since our investigations suggested that CHPs on NF-1 are not pre-assembled into triple helix due to spatial separation, we questioned whether the CHPs can form triple helical conjugates with exogenously supplied free CHPs. We coated a 96-well ELISA plate with NF-1, nanofibers made from (FKFE)2 conjugated to a scrambled CHP sequence (NF-1S) (Figure S7 and Table 1) and (FKFE)2 nanofibers without any CHP (designated as control nanofiber, NF-C) (Figure S6 and Table 1). The plate was treated with carboxyfluorescein (CF) labelled CHPs; either CF-G3-(GPO)9 (CF-CHP) or a CHP with a scramble sequence as a control (CF-CHP-S). The free peptides were heated at 80 °C for 5 min to completely disrupt any triple helical structures before applying to the plate coated with the nanofibers. Among all combinations tested, binding only occurred between CF-CHP and the NF-1 coated wells, strongly indicating that the CHP binds to NF-1 by triple helical hybridization (Figure 5A). In addition to utilizing NF-1 to target dColl for disease detection, these results suggest that NF-1’s capacity to stably capture free CHPs could potentially be used to help further functionalize the fibers with other bioactive molecules or detection probes. CHP

was

previously

shown

to

bind

to

dColl

by

triple

helical

hybridization,12,13,55,56 and we expected an increase in binding affinity for NF-1 due to the multi-ligand effect. Furthermore, since the dense layers of single strand CHPs on the fiber are separated at fixed distance which prevent self-interaction, we also expected that no pre-conditioning, such as heating, would be needed to activate NF-1 prior to dColl binding. We coated the ELISA plate with a thin layer of gelatin (type I collagen),


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followed by application of CF labeled nanofibers (CF-NF-1, CF-NF-1S, and CF-NF-C) as well as CF labeled CHPs. Except for the heated CF-CHP, all other CHPs and nanofibers were given ample chance to fold into triple helix by storing at 4 °C for at least 1 week prior to application to the plate. The binding was first allowed to take place at 37 °C for 2 hr followed by overnight washing at the same temperature. CF-NF-1 exhibited binding affinity to dColl, even after overnight washing (Figure 5B), while the heated and non-heated CF-CHP as well as the scrambled and control nanofibers (CF-NF-1S, CFNF-C) had negligible binding. Furthermore, approximately 75% of CF-NF-1 that bound to the collagen within the first 2 hr still remained bound after 10 days of washing at 37 °C (Figure S8) demonstrating exceptional affinity to gelatin. CF-NF-1 that was incubated with (GPO)9 at 4 °C to saturate the nanofiber’s collagen binding sites showed a significantly reduced level of binding when compared to the untreated CF-NF-1 (Figure 5B), which further corroborated the triple helix mediated binding mechanism. The results demonstrate that even without any preconditioning such as heat treatment or UV irradiation, the NF-1 can bind to denatured collagen with high affinity. Despite having the (FKFE)2 domain which has both hydrophobic and charged amino acid residues, CF-NF-1 exhibited little non-specific binding against many common serum proteins in a simple ELISA assay (Figure S9). We believe that such low non-specific binding is made possible by the hydrophilic CHP layers that passivate the core β-sheet structure. This result is consistent with previously reported CHP conjugated nanoparticles which also have a dense layer of CHPs and were shown to be inert toward non-specific binding.14 To confirm the collagen specific binding of CF-NF-1, tissue sections taken from mouse skin were denatured with 80 °C water and further treated CF-



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NF-1 or CF-NF-1S (Figure 5C). Only CF-NF-1 was able to stain denatured collagen. The results confirm that the CF-NF-1 has specific affinity to denatured collagen and that the binding is mediated by triple helical hybridization. Despite high affinity for dColl, NF-1 cannot be used for in vivo targeting of collagens in the extracellular matrix (ECM) due to the large size which hinders diffusion into the tissue from systemic circulation. Therefore, we considered a way to control and possibly shorten the length of NF-1. The temperature dependent CD experiment (Figure S10) suggested that the secondary structure of NF-1 was altered when the temperature reached 80 °C as evidenced by significant changes in the shape of the CD trace. However, the CD trace at 80 °C still showed reminiscence of the peak at 202 nm corresponding to the aromatic π-π stacking interactions of the bilayer peptide assembly. Therefore, we reasoned that heating NF-1 could disrupt the hydrogen bonds between the bilayer peptide assembly without disrupting the bilayer structure which could lead to breakdown of the long fibers into short fibers. To test this idea, we subjected NF-1 (2 mM in water) to predetermined temperatures between 80 to 100 °C. We found that the lengths of NF-1 were considerably reduced from over 1 µm (Figure 3) before the heat treatment down to approximately 100 nm (Figure 6A-C) after the heat treatment. When observed under TEM, the length of NF-1 treated at 80 °C for 5 min was 203 ± 43 nm, while treatment at 90 °C for 5 min further reduced the length to 128 ± 36 nm. We did not observe any drastic change (107 ± 29 nm) when the sample was treated at 100 °C for 10 min. Moreover, the shortened length after the heat treatment remained unchanged even after incubation at either 4 °C (data not shown) or at room temperature for up to 1 month (Figure 6D-F).


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It is known that the antiparallel β-sheet fiber elongates along the fiber axis by the antiparallel β-sheet pairs connecting via hydrogen bonds.44 Thus, we believe that the long NF-1 was fragmented into shorter fibers under high temperature treatment by the disruption of the hydrogen bonds in the (FKFE)2 backbone. Although much progress has been made in the field of self-assembled peptide nanofibers,26,44,47,48,57,58 there is only a few reported methods for controlling the fiber length, such as adjusting the pH condition,44 or templating the peptide.59,60 We believe that our discovery of NF-1 and the simple thermal procedure to control the length of the fiber are exciting findings in the field of peptide self-assembly, however, further investigation is needed to fully elucidate the fragmentation mechanism and stabilization of the fragmented NF-1 made possible by the CHP domains. After successful production of short length NF-1, we investigated in vivo targeting behaviour of NF-1 for the dColl in the skeletal tissue with the help of near infrared fluorescence (NIRF) imaging as we have previously reported.13 Due to the high collagen remodelling activity, bones and cartilages in animals have elevated levels of dColl under normal conditions, which can be specifically targeted by CHPs via intravenous injection.13 Conjugation of near infrared dye (IR680) to (GPO)9-G3-(FKFE)2 resulted in formation of nanofibers (designated as IR-NF-1) with low water solubility. To overcome the solubility problem, which was mainly caused by the hydrophobic IR680 dye, we dissolved IR-NF-1 and NF-1 in hexafluoroisopropanol (HFIP) followed by mixing them to a final mole ratio of 1 to 4, respectively. Lyophilization of HFIP and addition of water yielded water-soluble nanofibers (designated as IR´-NF-1) that incorporated the IR680 conjugated peptide. As a control, we similarly produced water



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soluble IR680 conjugated nanofibers displaying scrambled CHP sequence (IR´-NF-1S). The resulting nanofibers were heated at 80 °C for 5 min to shorten the fiber length down to approximately 200 nm (Figure S11), and their affinity to denatured collagen was confirmed (Figure S12). All nanofibers and CHP samples were stored at 4 °C for at least a week prior to tail vein injection into normal mice. IR´-NF-1 targeted dColls in the skeletal tissues of normal mice with improved efficiency when compared to IR-CHP, as evidenced by the whole-body fluorescence images after the tail vein injection (Figure 7). For IR´-NF-1 treated mice, the images showed the uptake of nanofibers in the skeleton, while mice treated with the control samples, IR´-NF-1S and non-heated IR-CHP, did not show any skeletal uptake, with the signals clearing by 48 hr post injection (PI) (Figure 7A). As seen in Figure 7B, the skeleton of euthanized mice at 48 hr PI showed accumulation of IR´-NF-1 but not the IR´-NF-1S, particularly in the spine, ribs and joints (Figure 7B and Figure S13), which is comparable to the heated IR-CHP that we reported previously (Figure 7, IR-CHP heat).13 Surprisingly, there was no IR´-NF-1 intensity from the skin despite the IR-CHP injected mice having relatively high intensity in the skin (Figure 7, IR-CHP heat). This suggests that the IR´-NF-1 has higher specificity to collagens in the bone compared to IR-CHP which targets bone and skin both of which are known to harbor dColls resulting from high collagen remodeling activity.13 Recent reports demonstrated that molecular size larger than 70 kDa may not readily pass through the blood vessels of mouse skin under homeostatic conditions,61 alluding to the possibility that the endothelial barrier may have played a role in blocking IR´-NF-1 from accessing the remodeling collagens in the skin.61,62 It is also possible that the size of IR´-NF-1, which is orders of magnitude larger


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than IR-CHP, made it difficult to diffuse into the ECM of the skin and locate dColl. Additionally, blood vessels in the bone maintain large diameter with high permeability for cellular trafficking of bone marrow,63 which may have facilitated IR´-NF-1’s access to collagen in the bone ECM. Since there are a number of serious diseases associated with skeletal system, such as osteoporosis, arthritis, and bone metastasis, IR´-NF-1 could be further developed into new vehicle for bone targeted therapies. The high fluorescence intensity in the liver suggests that IR´-NF-1’s mechanism of clearance is by metabolic processing rather than renal clearance (Figure S14) which is understandable due to its large size. This is in drastic contrast to IR-CHP which showed no intensity in the liver, and moderate intensity in the kidney. IR´-NF-1S which did not bind to skeletal tissue seemed to have also cleared from the system by hepatic process as evidenced by the moderate intensity in the liver (Figure S15). IR´-NF-1’s signal in the lung was unexpected and needs further study to validate whether it is the result of collagen targeting or passive accumulation. Full toxicity and biodistribution studies are needed if IR´-NF-1 is to be developed into drug delivery vehicle; however the current results suggest that non-collagen bound, circulating nanofibers are cleared by the liver fairly quickly without accumulation.

Conclusions We produced a new type of nanofiber (NF-1) by self-assembling a peptide that incorporates both β-sheet and triple helical motifs. The β-sheet motifs contributed to the formation of nanofibers via beta-sheet bilayer packing, while the CHPs, which decorate the surface of the nanofibers, conferred high water solubility allowing for the formation



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of biochemically inert and non-aggregating nanofibers in aqueous solution. Although the CHPs are densely displayed on the nanofiber surface, they do not trimerize due to the fixed distance that physically separates the CHPs. This is a new concept in supramolecular assembly in which the principles of anti-parallel beta-sheet folding and fiber propagation are used to present high density of bioactive molecules on fiber surface with controlled orientation and inter-molecular spacing. NF-1 can non-covalently capture molecules with simple (GPO)6~9 sequence providing facile means for further functionalization. The high density of single strand CHPs gave nanofibers the exceptional affinity to dColl by multi-valency effect. By co-assembling of (FKFE)2 derivatives, as demonstrated in the making of IR´-NF-1, we can readily control the CHP density on the nanofibers or display other molecular probe on the fiber surface. We also demonstrated that the length of NF-1 can be shortened down to 100 nm, and when injected into normal mice, these short nanofibers can specifically target dColls in the skeletal tissues with little partition into dermal tissue. We envision using these nanofibers for detecting collagen fragments associated with disease or for theranostic targeting of collagens in the microenvironment of diseased tissues.

Methods Peptide synthesis: All peptides were synthesized on a Focus XC peptide synthesizer (AAPPTec, Louisville, KY) using TentaGel R RAM resins and standard F-moc chemistry with HBTU/DIEA (Advanced ChemTech, Louisville, KY) activation. Cleaved peptides were purified by reverse phase high performance liquid chromatography (HPLC) (Agilent, Santa Clara, CA) on a C18 column with a gradient of water-acetonitrile


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mixture (from 5 to 50%) containing 0.1% trifluoroacetic acid. The mass and purity of the peptides were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (UltrafleXtreme, Bruker Daltonics, Billerica, MA) and HPLC. Concentration of each peptide solution was calculated from the weight of dry peptide powder. IR680 dye (Li-COR, Lincoln, NE) and 5(6)-carboxyfluorescein (CF) (Sigma-Aldrich, St. Louis, MO) conjugated peptides were synthesized as reported before.13 All peptides (Table 1) were dissolved in DI water (unless specified otherwise) to yield a final concentration of 2 mM, and stored at 4 °C for further characterization.

Nanofiber assembly: Lyophilized peptide powder was first dissolved in water to make a 2 mM solution, followed by incubating at 80 °C for 1 min. This solution was cooled down to 4 °C and kept at this temperature for at least 2 hr to assemble the nanofibers. Identical nanofibers can be also produced by slowly cooling the 80 °C solution to room temperature.

Transmission electron microscopy (TEM): TEM was performed on a FEI Tecnai T12 microscope (FEI, Hillsboro, OR) operated at 120 kV. TEM samples were prepared by applying a drop of nanofiber solution onto a copper grid covered with a thin carbon film (EMS, Hatfield, PA), followed by staining with 2% uranyl acetate, and overnight drying at room temperature. The average length of the nanofibers was calculated from at least 100 different nanofibers. The images were processed using Gatan Digital Micrograph software (Gatan, Pleasanton, CA) and ImageJ (National Institutes of Health, Bethesda, MD).



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Atomic force microscopy (AFM): AFM was obtained on a Bruker Dimension Icon (Bruker, Billerica, MA). Nanofiber solution (5 µL) was pipetted onto a freshly cleaved mica substrate, followed by quick air-drying. Samples were scanned in air under tapping mode and images were processed using Nanoscope Analysis (Bruker, Billerica, MA).

Circular dichroism (CD): CD spectra were recorded on a JASCO J-1500 CD (JASCO, Tokyo) in a 0.10 mm quartz cell. All peptide samples were prepared in water and incubated at 4 °C overnight prior to CD measurement. Spectra were recorded from 190 to 250 nm with 0.5 nm increments at a scanning rate of 100 nm/min. CD melting experiments were performed in the temperature range from 20 °C to 90 °C at a heating rate of 1 °C/min. The intensity of the CD signal at 225 nm was monitored as a function of temperature. Raw CD signal was normalized by the concentration and by the number of residues.13 Melting temperatures were determined from the maximum of the first derivative of the melting curves.

Fourier transform infrared spectroscopy (FTIR): FTIR spectroscopy was acquired using a Nicolet iZ10 Fourier transform infrared spectrometer (Thermo Scientific, Grand Island, NY). Nanofiber solution (500 µL, 2 mM) was lyophilized, and the peptide powder and potassium bromide were mixed and compressed into a thin pellet. Data were fitted using Gaussian function.

X-ray diffraction (XRD): (FKFE)2 and (GPO)9-G3-(FKFE)2 peptides were dissolved in


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DI water to yield nanofiber solutions as described above. They were dried and packed into MicroRT™ Capillaries (MiTeGen, Ithaca, NY). The x-ray diffraction patterns were collected at room temperature on a Rigaku Micromax-007 x-ray generator (Rigaku, Woodlands, TX) equipped with an R-Axis IV++ area detector. The sample to detector distance was 200 mm. Each diffraction pattern was collected for 60 sec. For background subtraction, diffraction pattern was also acquired without the sample-containing capillary. Multiple images (ten images for each sample) were combined to improve the signal to noise ratio. One dimensional intensity profiles (intensity vs d-spacing) were obtained from the 2D XRD pattern using FIT2D.64 Microcal Origin (OriginLab, Northampton, MA) was used for peak analysis of the 1D x-ray data.

Gelatin binding assay: Gelatin binding assay was performed using an ELISA plate. Gelatin solution (type I collagen, 50 µL, 0.5 mg/mL) was added to each well in the ELISA plate and the plate was gently shaken at room temperature for 2 hr. The plate was washed three times with 1× PBS buffer, followed by blocking with 5% BSA at room temperature for 1 hr, and washing with PBS buffer three times. Aqueous solution of CFNF-1, (50 µL, 10 µM) was added to dColl coated plate and incubated at 37 °C for 2 hr, followed by washing three times with PBS buffer at 37 °C. SpectraMax M-2 microplate reader (Molecular Devices, Sunnyvale, CA) was used to measure the fluorescence (ex: 489 nm, em: 533 nm, cutoff: 530 nm). To saturate the binding sites of CF-NF-1, an excess amount of single strand CHP solution (100 µL, 400 µM) was added to the CF-NF1 solution (100 µL, 20 µM) and incubated at 4 °C overnight. Each binding experiment was performed in triplicate.



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Histological tissue staining: Frozen skin tissue sections obtained from a normal mouse were treated three times with 1 mL of boiling water to induce thermal burn, followed by cooling to room temperature prior to staining. CF-NF-1 or CF-NF-1S (100 µL, 15 µM) was applied to the tissue section. The tissue sections were incubated in a humidity chamber at 4 °C overnight, followed by rinsing in PBS for 5 min, three times. An anticollagen antibody (Abcam, 1 mg/mL) diluted to 1:100 was applied to the tissue section at 4 °C for at least 2 hr, stained with Alexa Fluor555-labeled donkey anti-rabbit IgG H&L (1:200 dilution) for 1 hr, followed by staining with Hoechst for 20 min at room temperature and final rinsing before mounting on a microscope. The images were acquired using an EVOS FL fluorescence microscopy (Thermo Fisher Scientific, Waltham, MA) with a 20× objective lens.

Animal study: All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines of the University of Utah. Female Nu/Nu mice at the age of 5-6 weeks were used in this study. The tail vein injection was performed using 2 nmol of IR680 conjugated CHP nanofibers with a maximum injection volume of 200 µL. Mice were visualized at predetermined time points post-injection (30 min, 6 hr, 24 hr, and 48 hr) using an IVIS imager (PerkinElmer, Waltham, MA) with an excitation/emission wavelength of 675/720 nm and an exposure time of 1 sec. Mice were sacrificed at 48 hr time point. Organs and skeletons were collected for further analysis and scanned using the same imager. N = 5 for all studies.


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Associated content Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: CD, TEM, FTIR, and XRD of the nanofibers; Specificity of NF-1 binding to denatured collagen; NIRF images of denatured collagens targeted in vivo.

Author information Corresponding author. Email: [email protected] Notes Drs. Yu and Li are founders of 3Helix which commercializes collagen hybridizing peptide. Acknowledgement This work was supported by grants from NIAMS/NIH (R01-AR060484 and R21AR065124) and DOD (W81XWH-12-1-0555) awarded to S.M.Y. We thank Drs. Christopher P. Hill and Frank Whitby for access to the x-ray diffraction facility.

Reference (1)

Badylak, S. F.; Freytes, D. O.; Gilbert, T. W. Acta Biomater. 2009, 5, 1.

(2)

Jin, H.-E.; Farr, R.; Lee, S.-W. Biomaterials 2014, 35, 9236.

(3)

Federico, S.; Pierce, B. F.; Piluso, S.; Wischke, C.; Lendlein, A.; Neffe, A. T. Angew. Chem. Int. Ed. 2015, 54, 10980.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(4)

Page 30 of 43

Rothenfluh, D. A.; Bermudez, H.; O/'Neil, C. P.; Hubbell, J. A. Nat. Mater. 2008, 7, 248.

(5)

Chan, J. M.; Zhang, L.; Tong, R.; Ghosh, D.; Gao, W.; Liao, G.; Yuet, K. P.; Gray, D.; Rhee, J.-W.; Cheng, J.; Golomb, G.; Libby, P.; Langer, R.; Farokhzad, O. C. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 2213.

(6)

Stuart, K.; Paderi, J.; Snyder, P. W.; Freeman, L.; Panitch, A. PLoS ONE 2011, 6, e22139.

(7)

Paderi, J. E.; Panitch, A. Biomacromolecules 2008, 9, 2562.

(8)

Hunter, G. K.; Poitras, M. S.; Underhill, T. M.; Grynpas, M. D.; Goldberg, H. A. J. Biomed. Mater. Res. 2001, 55, 496.

(9)

Helms, B. A.; Reulen, S. W. A.; Nijhuis, S.; Graaf-Heuvelmans, P. T. H. M. d.; Merkx, M.; Meijer, E. W. J. Am. Chem. Soc. 2009, 131, 11683.

(10)

Wahyudi, H.; Reynolds, A. A.; Li, Y.; Owen, S. C.; Yu, S. M. J. Control. Release 2016, 240.

(11)

Bonnans, C.; Chou, J.; Werb, Z. Nat. Rev. Mol. Cell Biol. 2014, 15, 786.

(12)

Li, Y.; Yu, S. M. Curr. Opin. Chem. Biol. 2013, 17, 968.

(13)

Li, Y.; Foss, C. A.; Summerfield, D. D.; Doyle, J. J.; Torok, C. M.; Dietz, H. C.; Pomper, M. G.; Yu, S. M. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 14767.

(14)

San, B. H.; Li, Y.; Tarbet, E. B.; Yu, S. M. ACS Appl. Mater. Interfaces 2016, 8, 19907.

(15)

Stahl, P. J.; Chan, T. R.; Shen, Y.-I.; Sun, G.; Gerecht, S.; Yu, S. M. Adv. Funct. Mater. 2014, 24, 3213.


Page 31 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(16)

Zitnay, J. L.; Li, Y.; Qin, Z.; San, B. H.; Depalle, B.; Reese, S. P.; Buehler, M. J.; Yu, S. M.; Weiss, J. A. Nat. Comm. 2017, 8, 14913.

(17)

Hwang, J.; San, B. H.; Turner, N. J.; White, L. J.; Faulk, D. M.; Badylak, S. F.; Li, Y.; Yu, S. M. Acta Biomat. 2017, 53, 268.

(18)

Shoulders, M. D.; Guzei, I. A.; Raines, R. T. Biopolymers 2008, 89, 443.

(19)

Hodges, J. A.; Raines, R. T. J. Am. Chem. Soc. 2005, 127, 15923.

(20)

Boudko, S.; Frank, S.; Kammerer, R. A.; Stetefeld, J.; Schulthess, T.; Landwehr, R.; Lustig, A.; Bächinger, H. P.; Engel, J. J. Mol. Biol. 2002, 317, 459.

(21)

Ackerman, M. S.; Bhate, M.; Shenoy, N.; Beck, K.; Ramshaw, J. A. M.; Brodsky, B. J. Biol. Chem. 1999, 274, 7668.

(22)

Li, Y.; Ho, D.; Meng, H.; Chan, T. R.; An, B.; Yu, H.; Brodsky, B.; Jun, A. S.; Michael Yu, S. Bioconj. Chem. 2013, 24, 9.

(23)

Li, Y.; San, B. H.; Kessler, J. L.; Kim, J. H.; Xu, Q.; Hanes, J.; Yu, S. M. Macromol. Biosci. 2015, 15, 52.

(24)

Shoulders, M. D.; Raines, R. T. Annu. Rev. Biochem. 2009, 78, 929.

(25)

Santos, J. L.; Li, Y.; Culver, H. R.; Yu, M. S.; Herrera-Alonso, M. Chem. Comm. 2014, 50, 15045.

(26)

O'Leary, L. E. R.; Fallas, J. A.; Bakota, E. L.; Kang, M. K.; Hartgerink, J. D. Nat. Chem. 2011, 3, 821.

(27)

Rele, S.; Song, Y.; Apkarian, R. P.; Qu, Z.; Conticello, V. P.; Chaikof, E. L. J. Am. Chem. Soc. 2007, 129, 14780.

(28)

Sarkar, B.; O’Leary, L. E. R.; Hartgerink, J. D. J. Am. Chem. Soc. 2014, 136, 14417.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(29)

Tanrikulu, I. C.; Forticaux, A.; Jin, S.; Raines, R. T. Nat. Chem. 2016, 8, 1008.

(30)

Jiang, T.; Xu, C.; Liu, Y.; Liu, Z.; Wall, J. S.; Zuo, X.; Lian, T.; Salaita, K.; Ni,

Page 32 of 43

C.; Pochan, D.; Conticello, V. P. J. Am. Chem. Soc. 2014, 136, 4300. (31)

Jiang, T.; Xu, C.; Zuo, X.; Conticello, V. P. Angew. Chem. Int. Ed. 2014, 53, 8367.

(32)

Przybyla, D. E.; Chmielewski, J. J. Am. Chem. Soc. 2010, 132, 7866.

(33)

Gleaton, J.; Chmielewski, J. ACS Biomater. Sci. Eng. 2015, 1, 1002.

(34)

Kojima, C.; Tsumura, S.; Harada, A.; Kono, K. J. Am. Chem. Soc. 2009, 131, 6052.

(35)

Kojima, C.; Suehiro, T.; Tada, T.; Sakamoto, Y.; Waku, T.; Tanaka, N. Soft Matter 2011, 7, 8991.

(36)

Luo, T.; Kiick, K. L. Eur. Polym. J. 2013, 49, 2998.

(37)

Luo, T.; Kiick, K. L. J. Am. Chem. Soc. 2015, 137, 15362.

(38)

Measey, T. J.; Markiewicz, B. N.; Gai, F. Chem. Phys. Lett. 2013, 580, 135.

(39)

Bowerman, C. J.; Nilsson, B. L. J. Am. Chem. Soc. 2010, 132, 9526.

(40)

Rudra, J. S.; Sun, T.; Bird, K. C.; Daniels, M. D.; Gasiorowski, J. Z.; Chong, A. S.; Collier, J. H. ACS Nano 2012, 6, 1557.

(41)

Lim, Y.-b.; Kwon, O.-J.; Lee, E.; Kim, P.-H.; Yun, C.-O.; Lee, M. Org. Biomol. Chem. 2008, 6, 1944.

(42)

Lee, N. R.; Bowerman, C. J.; Nilsson, B. L. Biomacromolecules 2013, 14, 3267.

(43)

Engel, J.; Bächinger, H. P. In Collagen: Primer in Structure, Processing and Assembly; Brinckmann, J., Notbohm, H., Müller, P. K., Eds.; Springer Berlin Heidelberg: Berlin, Heidelberg, 2005, p 7.


Page 33 of 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(44)

Dong, H.; Paramonov, S. E.; Aulisa, L.; Bakota, E. L.; Hartgerink, J. D. J. Am. Chem. Soc. 2007, 129, 12468.

(45)

Bella, J.; Eaton, M.; Brodsky, B.; Berman, H. M. Science 1994, 266, 75.

(46)

Marini, D. M.; Hwang, W.; Lauffenburger, D. A.; Zhang, S.; Kamm, R. D. Nano Lett. 2002, 2, 295.

(47)

Koutsopoulos, S.; Unsworth, L. D.; Nagai, Y.; Zhang, S. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 4623.

(48)

Kumar, V. A.; Taylor, N. L.; Shi, S.; Wang, B. K.; Jalan, A. A.; Kang, M. K.; Wickremasinghe, N. C.; Hartgerink, J. D. ACS Nano 2015, 9, 860.

(49)

Li, Y.; Wang, F.; Cui, H. Bioengineering & Transla. Med. 2016, 1, 306.

(50)

Rughani, R. V.; Branco, M. C.; Pochan, D. J.; Schneider, J. P. Macromolecules 2010, 43, 7924.

(51)

Branco, M. C.; Pochan, D. J.; Wagner, N. J.; Schneider, J. P. Biomaterials 2009, 30, 1339.

(52)

Caplan, M. R.; Moore, P. N.; Zhang, S.; Kamm, R. D.; Lauffenburger, D. A. Biomacromolecules 2000, 1, 627.

(53)

Caplan, M. R.; Schwartzfarb, E. M.; Zhang, S.; Kamm, R. D.; Lauffenburger, D. A. Biomaterials 2002, 23, 219.

(54)

Zhang, S.; Holmes, T.; Lockshin, C.; Rich, A. Proc. Natl. Acad. Sci. U. S. A. 1993, 90, 3334.

(55)

Wang, A. Y.; Foss, C. A.; Leong, S.; Mo, X.; Pomper, M. G.; Yu, S. M. Biomacromolecules 2008, 9, 1755.

(56)

Wang, A. Y.; Mo, X.; Chen, C. S.; Yu, S. M. J. Am. Chem. Soc. 2005, 127, 4130.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(57)

Cui, H.; Muraoka, T.; Cheetham, A. G.; Stupp, S. I. Nano Lett. 2009, 9, 945.

(58)

Ulijn, R. V.; Woolfson, D. N. Chem. Soc. Rev. 2010, 39, 3349.

(59)

Pal, A.; Malakoutikhah, M.; Leonetti, G.; Tezcan, M.; Colomb-Delsuc, M.;

Page 34 of 43

Nguyen, V. D.; van der Gucht, J.; Otto, S. Angew. Chem. Int. Ed. 2015, 54, 7852. (60)

Bull, S. R.; Palmer, L. C.; Fry, N. J.; Greenfield, M. A.; Messmore, B. W.; Meade, T. J.; Stupp, S. I. J. Am. Chem. Soc. 2008, 130, 2742.

(61)

Egawa, G.; Nakamizo, S.; Natsuaki, Y.; Doi, H.; Miyachi, Y.; Kabashima, K. Sci. Rep. 2013, 3, 1932.

(62)

Pasparakis, M.; Haase, I.; Nestle, F. O. Nat. Rev. Immunol. 2014, 14, 289.

(63)

Itkin, T.; Gur-Cohen, S.; Spencer, J. A.; Schajnovitz, A.; Ramasamy, S. K.; Kusumbe, A. P.; Ledergor, G.; Jung, Y.; Milo, I.; Poulos, M. G.; Kalinkovich, A.; Ludin, A.; Kollet, O.; Shakhar, G.; Butler, J. M.; Rafii, S.; Adams, R. H.; Scadden, D. T.; Lin, C. P.; Lapidot, T. Nature 2016, 532, 323.

(64)

Hammersley, A., ESRF Internal Report, ESRF97HA02T, 1997, "FIT2D: An Introduction and Overview."


Page 35 of 43

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Figures and Figure Legends

Figure 1. Schematic of CHP nanofiber self-assembly. The nanofiber forming CHP (NF1) has a sequence of (GPO)9-G3-(FKFE)2, where antiparallel β-sheet forming segment, (FKFE)2 is linked to the CHP sequence, (GPO)9 via a G3 linker (left panel). This peptide self-assembles into a nanofiber with a left handed twist by forming a core of extended anti-parallel β-sheet bilayer which displays dense layer of single strand CHPs with precisely defined interchain spacing (right panel, top and bottom β-sheet layers are colored in black and grey, respectively). According to x-ray diffraction, the distance between the two adjacent β-strands within the β-sheet is 4.6 Å, and the distance between the two β-sheets forming the bilayer is 11.2 Å. The calculated overall width of the nanofiber is 20.4 nm. The direction of the thin arrows indicate the polarity (C to N terminus) of the peptide. The two thick black arrows pointing up and down indicate the hydrogen bond-mediated propagation direction of the nanofiber.



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Figure 2. CD measurements of CHP nanofibers. (A) CD profiles of (GPO)9-G3-(FKFE)2 nanofiber (NF-1), (FKFE)2 nanofiber (NF-C), and (GPO)9. The spectrum of (GPO)9-G3(FKFE)2 after subtracting the (GPO)9 trace is also shown (double dot dashed line). (B) CD melting curves of NF-1 (solid line) and (GPO)9 (dashed line).


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Figure 3. TEM (A) and AFM (B) of NF-1. AFM height profile (C) corresponds to the marked area of the nanofiber in the inset of B.



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Figure 4. X-ray diffraction of NF-1. Arrows indicate two major diffraction peaks corresponding to the 4.6 Å spacing (grey arrow) between the two adjacent β-strands within the β-sheet, and the 11.2 Å spacing (black arrow) between the top and bottom layers of the bilayer.


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Figure 5. NF-1 binding specifically to denatured collagen. (A) NF-1, NF-C and NF-1S were coated on an ELISA plate, followed by application of either the fluorescently labeled CHP (CF-CHP) or fluorescently labeled CHP with a scrambled sequence (CFCHP-S). (B) The CHPs and nanofibers were allowed to bind to denatured collagencoated ELISA plate at 37 °C for 2 hr, followed by overnight washing at 37 °C. The heated CF-CHPs were heated at 80 °C for 5 min to melt any triple helical structure before use. (C) Histological tissue staining of burn skin model. Frozen section of mouse skin previously treated with hot water was stained with CF-NF-1 (green), anti-collagen type I antibody (anti-Col I Ab, red), and Hoechst (nucleus, blue). Only CF-NF-1 binds to the tissue sections. Scale bar: 100 µm.



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Figure 6. TEM of heat treated NF-1. Micrographs and corresponding histograms of fiber lengths are shown for 2 mM NF-1 solution which was heated at 80 °C for 5 min (A); at 90 °C for 5 min (B); at 100 °C for 10 min (C). D, E, and F correspond to the solutions A, B, and C stored at room temperature for 1 month, respectively. Scale bar: 200 nm.


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Figure 7. In vivo targeting of denatured collagens in skeletal tissues. (A) Whole body NIRF images of normal Nu/Nu mice injected intravenously with IR´-NF-1, IR´-NF-1S, or IR-CHP (heat or non-heat) at noted time points post-injection (PI). (B) NIRF images of skeletal and skin tissues isolated from corresponding mice 48 hr PI. The heated IRCHP was heated at 80 °C for 5 min to melt the triple helices to generate single strand CHPs, followed by quenching on ice for 30 sec before injection.



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Table 1. Peptide Sequence and Molecular Weight Peptide

Sequence

Calculated [M+H]+ 3695.8 1120.6 3695.8 4166.1 1589.9 4166.1 4699.4 4653.2 2422.2 2950.5 2950.5 3379.6

NF-1 (GPO)9-G3-(FKFE)2 NF-C (FKFE)2 NF-1S (FKFE)2-G3-G9P9O9 CF-NF-1 CF-Ahx-(GPO)9-G3-(FKFE)2 CF-NF-C CF-Ahx-(FKFE)2 CF-NF-1S CF-Ahx-(FKFE)2-G3-G9P9O9 IR-NF-1 IR680-PEG-(GPO)9-G3-(FKFE)2 IR-NF-1S IR680-Ahx-(FKFE)2-G3-G9P9O9 CHP (GPO)9 CF-CHP CF-G3-(GPO)9 CF-CHP-S CF-G3-G9P9O9 IR-CHP IR680-G3-(GPO)9 Ahx: aminohexanoic acid G9P9O9: PGOGPGPOPOGOGOPPGOOPGGOOPPG

MALDI-TOF [M]+ 3696.1 1120.6 3695.5 4168.8 1591.8 4167.1 4700.3 4655.3 2423.2 2952.5 2952.5 3379.8


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Table of Content graphic


Self-Assembled Water-Soluble Nanofibers Displaying Collagen

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