WO2018106273A1 - Collagen targeting nanofibers and nanosheets - Google Patents

Collagen targeting nanofibers and nanosheets Download PDF

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Publication number
WO2018106273A1
WO2018106273A1 PCT/US2017/019724 US2017019724W WO2018106273A1 WO 2018106273 A1 WO2018106273 A1 WO 2018106273A1 US 2017019724 W US2017019724 W US 2017019724W WO 2018106273 A1 WO2018106273 A1 WO 2018106273A1
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Prior art keywords
peptide
collagen
peptide conjugate
seq
gpo
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PCT/US2017/019724
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French (fr)
Inventor
S. Michael YU
Yang Li
Boi Hoa SAN
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University Of Utah Research Foundation
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Publication of WO2018106273A1 publication Critical patent/WO2018106273A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]

Definitions

  • Collagen hybridizing peptide has an extremely strong tendency to self- assemble into a triple helix structure.
  • collagen hybridizing peptide needs to present in single strand or unfolded form. So far, strategies for either avoiding collagen hybridizing peptide self-trimerization or for collagen hybridizing peptides readily binding to denatured collagen are not well- developed, even though the use of these collagen hybridizing peptide-conjugated materials for many applications, including imaging, drug delivery, and tissue engineering can be crucial.
  • the first strategy is to use heat treatment, since collagen hybridizing peptides are melted when they are heated above their melting temperatures.
  • the former strategy using heat activated collagen hybridizing peptide seems to be ideal for collagen hybridizing peptide-based applications, especially in vivo therapeutics, because it eliminates potential toxicity of the photo-cleaved products and also simplifies the collagen hybridizing peptide structures for easy conjugation to other bioactive molecules. Even though both methods can generate single strand collagen hybridizing peptides, they still self-reassemble into triple helical structures when they are below their melting
  • peptide conjugates comprising a (FKFE) 2 (SEQ ID NO: 1) beta- sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
  • peptide conjugates comprising a (FKFE) 2 (SEQ ID NO: 1) beta- sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide, wherein the collagen hybridizing peptide comprises the sequence of at least (GXY)n, wherein G is glycine, wherein X and Y are any amino acid, and wherein n is at least 3.
  • compositions comprising one or more of the disclosed peptide conjugates.
  • compositions comprising one or more of the peptide conjugates comprising a (FKFE) 2 (SEQ ID NO: l) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
  • nanofibers comprising two or more of the disclosed peptide conjugates.
  • nanofibers comprising two or more of the peptide conjugates comprising a (FKFE) 2 (SEQ ID NO: l) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
  • nanosheets comprising two or more of the disclosed nanofibers.
  • nanosheets comprising two or more of the nanofibers comprising two or more of the peptide conjugates comprising a (FKFE) 2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
  • FKFE FKFE 2
  • Disclosed are methods of detecting denatured collagen in a sample comprising contacting a composition comprising the one of the disclosed peptide conjugates, and detecting the presence or absence of binding of the peptide conjugate to denatured collagen, wherein the presence of binding indicates the presence of denatured collagen.
  • a peptide conjugate comprising a (FKFE) 2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide can be contacted with a sample.
  • peptide conjugates comprising a (FKFE) 2 (SEQ ID NO: l) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide can be administered.
  • Disclosed are methods of screening for a therapeutic agent that treats a disease or injury involving collagen damage comprising detecting the presence or absence of binding of a peptide conjugate to denatured collagen, the presence of binding indicating the presence of denatured collagen in the sample; administering to a sample having denatured collagen a composition comprising a peptide conjugates comprising a (FKFE) 2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide, wherein the active agent is a detectable moiety; and detecting the presence or absence of binding of the peptide conjugate to denatured collagen, the presence of binding indicating the presence of denatured collagen in the sample, wherein a decrease in the amount of damaged or denatured collagen in the sample detected after administration of the peptide conjugate compared to the amount detected prior to administration of the peptide conjugate indicates a therapeutic agent capable of a disease or injury involving collagen damage.
  • FKFE FKFE
  • kits comprising a (FKFE) 2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide. Also disclosed are kits comprising a peptide conjugate comprising a (FKFE) 2 (SEQ ID NO: l) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
  • Nanofibers comprise of a CHP sequence of (GPO)9, (SEQ ID NO: 5), a linker G 3 , and an anti-parallel ⁇ -sheet forming peptide (FKFE) 2 (SEQ ID NO: 1).
  • the two nanofibers When the two nanofibers are mixed together, they assemble into a single layer nanosheet (designated as NS) by triple helical folding of parallely-aligned CHPs.
  • the blue and green arrows indicate the elongated direction of NF-1 and NF-2, respectively, as well as in the case of NS.
  • FIGS 2A-2I Transmission electron microscopy (TEM) and atomic force microscopy (AFM) images of nanofibers and nanosheet.
  • A) and (B) are TEM and AFM of NF-1, respectively.
  • D) and (E) are TEM and AFM of NF-2, respectively.
  • G) and (H) are TEM and AFM of NS, respectively.
  • the blue arrows in each AFM indicate the width and the height where the measurements were taken.
  • the bottom panel (C, F, I) in each AFM is the corresponding height and width profile with the number indicating the average width of the nanofibers.
  • FIGS 3A-3B Gelatin binding property of nanofibers. 5(6)- carboxy fluorescein (CF) conjugated nanofibers were used to study the binding to gelatin.
  • A Ten ⁇ of each nanofiber was incubated with gelatin coated ELISA-type plate at 37 °C for 2 hours.
  • B Samples in (A) were washed at 37 °C overnight. NF-1 and NF-2 were pre-heated at 60 °C for 5 minutes to disrupt the potential intra-fiber CHP folding.
  • FKFE) 2 SEQ ID NO: 1 fiber alone and (FKFE) 2 (SEQ ID NO: 1) fiber with a CHP scrambled sequence (SEQ ID NO: 4) were used as controls.
  • FIG. 1 Denatured collagen (Gelatin) behavior study of CHP-(FKFE) 2 (SEQ ID NO: 1) nanofibers.
  • the nanofibers were coated on a ELISA-type plate, followed by testing their binding behaviors with fluorescent-labelled CF-(GPO)9 (SEQ ID NO:5) and scrambled sequence CF-G 9 P 9 O 9 (SEQ ID NO:6).
  • FIG. 6 is a schematic diagram of the basic structure of collagen is composed of a triple helix of 3 alpha chains.
  • the most common amino acid motifs of collagen are glycine-proline-hydroxyproline, every third residue should be a glycine.
  • the collagen mimetic peptide (CHP) is a synthetic peptide that mimics the basic structural motif of natural collagen— the triple helix, which shares the Gly-Pro-Hyp triplet.
  • the Collagen Mimetic Peptide has a strong propensity to self-assemble into a triple helical structure.
  • Figure 7 is a schematic diagram of a single-strand synthetic CHP with a sequence from 6 to 10 (GPO) repeats that can specifically bind to denatured collagens through a unique triple-helix hybridization mechanism, which is similar to DNA hybridization.
  • GPO 6 to 10
  • FIG 8 is a schematic diagram showing that CHP has many potential applications, however, the problem with CHPs is that they have a high propensity to self- trimerize to form triple helical structure, and this will significantly reduce their targeting efficacy.
  • the easy way to make CHP into single strand is to use heat to disrupt their triple helices.
  • Another way to make CHP stay in single strand is to introduce a photo-cleavable nitrobenzene (NB) group in CHP to prevent them from self-trimerizing by hindrance effect, in this case a NB group is placed in the middle of CHP sequence.
  • NB group photo-cleavable nitrobenzene
  • these strategies still cannot completely prevent them from self-trimerizing once they are cool down or the NB groups get removed by UV light.
  • the disclosed CHP system is unable to self trimerize but still can target denatured collagen without any precondition like heating or UV -light irradiating.
  • Figure 9 is a schematic diagram showing how to turn CHP into nanofibers and nanosheets so that they can readily bind to denatured collagen with high affinity.
  • CHP was linked to a short anti-parallel beta-sheet forming peptide, so that they can self- assemble into nanofibers and be able to bind to denatured collagen by multivalence effect.
  • Figures 10A, 10B, and IOC show the TEM and AFM images of the CHP-NF. No aggregation was seen.
  • the dimensions of the NF are 1.5 nm in height, 22 nm in width, and over one micrometer in length.
  • Figure 11 shows circular dichroism (CD) measurement to investigate more about CHP-NF's secondary structure, it was confirmed that they assembled into a double- layered anti-parallel ⁇ -sheet nanofibers. The interesting thing is that a triple helical signal was not seen from CHP in NF.
  • Figure 12 shows the Tm measurement for this CHP-NF, unlike CHP peptide only which has a clear melting transition at about 75° C, no melting transition for CHP- NFs was seen, indicating that CHPs on NF did not self trimerize.
  • Figure 13 is a graph showing a gelatin behavior study of CHP-(FKFE)2 nanofibers.
  • the nanofibers were coated on an ELISA-type plate, followed by testing their binding behaviours with fluorescent labeled CF-(GPO)9 (SEQ ID NO:5) and scrambled sequence CF-G 9 P 9 O 9 (SEQ ID NO:6).
  • Figure 14 shows a longer CHP sequence can be displayed on the nanofiber platform and still can keep them for not self trimerizing.
  • CHPs with sequences of (GPO) repeats up to 12 can be displayed. Again, no melting transitions were seen for CHPs in CD measurement, indicating that they did not form triple helical structures on the NFs.
  • longer CHP sequences can give strong binding affinity, and the Tm of (GPO)i2 (SEQ ID NO: 76) is up to more than 100° C, which is very difficult to disrupt their triple helices by heating, also their refolding rate is very quick.
  • Figure 15 shows the binding affinity of CHP-NFs to denatured collagen using a denatured collagen binding assay.
  • the ELISA type plate was first coated with denatured collagen or gelatin then fluorescently labeled CHP-NFs was applied on the wells at 37° C, for 2 h, followed by washing out the unbound NFs to check the remaining fluorescent signals.
  • CHP-NFs showed exceptionally strong binding to denatured collagen at 37° C as compared with CHP alone, this can be due to multivalence effect.
  • FIG. 16 shows that CHP-NFs were highly soluble in water and had no tendency to aggregate or bind to proteins other than denatured collagen due to the inertness and hydrophilicity of the CHP sequence. This is totally opposite with many other beta-sheet forming NFs, which normally tend to aggregate and form hydrogels.
  • FIG. 17A-D shows that that CHP-NFs also behaved very similar to gelatin in cell culture, which means that they have no cytoxicity to cell culture.
  • Figure 18 shows that the length of the CHP-NFs can be controlled.
  • the average length of the CHP-NFs is over 1 um.
  • the NF length can be controlled by heating them in different temperatures. When we heated the NFs at 80° C/5 min, the NF length reduced to 200 nm, at 90° C/5 min, it further reduced to 100 -150 nm. No further change at longer heating time. No change in length was seen for 1 month incubation at 4° C, RT, or even at 37° C.
  • Figure 19 is a schematic diagram for testing the binding affinity of CHP-NFs in a mouse model. An IR dye was conjugated to the CHP-NF and injected into mice.
  • FIG. 20 shows results in a mouse study.
  • An IR680 dye was conjugated to the CHP-NF, and a NF was used with a CHP scramble sequence as a control. Without preheating the CHP-NF, they can readily bind to remodeling collagen in the bone. After one week, the signal is still very high.
  • Figure 21 shows the CHP-nanosheet structure.
  • 2 opposite polarity CHP-NFs were synthesized and then mixed together.
  • these 2 different orientation NFs were able to form a sheet like structure, called the CHP- nanosheet.
  • CHPs need to be in the same orientation. Therefore, by displaying CHPs in opposite polarity on NFs, a CHP-nanosheet structure can be made, which is very unique.
  • Figures 22A-F show the TEM and AFM images of the CHP-nanosheet. 2 opposite polarity CHP nanofibers can form a monolayer CHP-nanosheet.
  • A) and (B) are TEM and AFM of NF-1, respectively.
  • C) and (D) are TEM and AFM of NF-2, respectively.
  • E) and (F) are TEM and AFM of NS, respectively.
  • Figure 23 shows the Tm for nanosheet. There is a clear melting transition for sample comprised NF-1 + NF-2. However, no melting transition was seen for the sample comprised of NF-1 + NF (NF with a CHP scramble sequence, used as a control), indicating that NF-1 and NF-2 are able to assemble into nanosheets via triple helical formation.
  • Figure 24 shows the TEM image of the nanosheet with NP on the surface, proving that the nanosheet can be easily functionalized with bioactive molecules, such as biotin.
  • Figure 25 shows the results of antibody binding to the nanosheet.
  • the nanosheet was modified with even larger biomolecules, for example, in this case the nanosheet was functionalized with antibody, by adding an antibody binding motif to the nanosheet.
  • Figure 26 shows that this antibody functionalized NS is active. It was used to stimulate T cell activity. Because, it is well known that stimulating T cell using a stiff surface is more efficient than any other known methods.
  • CD3 and CD28 antibodies were used to co-stimulate the T cells, and significant enhancement in cell activity was found as compared with the free antibodies, as evidenced by the IL-2 production. No significant difference was found between NF and NS.
  • FIG 27 shows that the nanosheet can cluster the T cells while stimulating the cells.
  • T cell therapy has become one of the most promising treatments for many diseases such as cancers, but there are still many challenges (such as off target effect, delivering inactive cells, etc.) that need to be overcome before this method can be widely applied on patients.
  • Figure 28 shows that in the control samples, no cell clusters were seen. Even though the NF can also promote the T cell activity it cannot cluster the cells.
  • Figure 29 shows that the nanosheet can also bind to denatured collagen while still binding to antibodies.
  • Figure 30 shows that T cells clustered by nanosheet can also bind to denatured collagen.
  • treating refers to partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular disease, disorder, and/or condition.
  • "treating" a disease or injury involving collagen damage can refer to reducing or eliminating the amount of damaged/denatured/diseased collagen.
  • Treatment can also be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
  • subject refers to the target of administration, e.g. an animal.
  • the subject of the disclosed methods can be a vertebrate, such as a mammal.
  • the subject can be a human.
  • the term does not denote a particular age or sex.
  • Subject can be used interchangeably with “individual” or "patient.”
  • Ranges may be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range- 1 from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise.
  • peptide conjugates comprising a (FKFE) 2 (SEQ ID NO: 1) beta- sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
  • peptide conjugates comprising a (FKFE) 2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide, further comprising an active agent.
  • the active agent can be a detectable moiety or a therapeutic agent.
  • a detectable moiety can be, but is not limited to, a fluorescent dye, radioactive isotope, magnetic bead, metallic bead, colloidal particle, near-infrared dye, or an electron-dense reagent.
  • the active agent can be attached to the N- terminal or C-terminal portion of the peptide conjugate.
  • the therapeutic agent can be a therapeutic known to treat a disease or injury involving collagen damage.
  • the therapeutic agent is an antibody, a peptide, a nucleic acid, or a chemical compound.
  • the disease or injury can be, but is not limited to, cancer, osteoporosis, arthritis, fibrosis, a chronic wound, myocardial infarct, atherosclerotic plaques, blood vessel injury and Failure (stroke), osteoarthritis, rheumatoid arthritis, osteoporosis, bone fracture/healing, Marfan syndrome, mechanical injury, sports medicine, e.g., rotator cuff tear, skin damage (photo, aging), skin cancer, skin inflammation, cornea injury & infection, corneal healing after LASIK, keratoconus, wet AMD, lung
  • ECM extracellular matrix
  • the disclosed (FKFE) 2 (SEQ ID NO: 1) beta-sheet forming peptide comprises an alternating partem of hydrophobic and hydrophilic amino acids.
  • (FKFE) 2 (SEQ ID NO: 1) beta-sheet forming peptides can self-assemble into nanofibers. In some instances, the nanofibers can form nanosheets.
  • the spacer moiety can comprise at least three glycines, aminohexanoic acid, gamma- Aminobutyric acid, (2-AMINOETHOXY)ACETIC ACID, 3-( amino)-3-(2-nitrophenyl)propionic acid or PEG.
  • the spacer moiety can be any length and can be any molecule that helps link a (FKFE) 2 (SEQ ID NO: 1) beta-sheet forming peptide and a collagen hybridizing peptide.
  • a spacer moiety can also be called a linker.
  • peptide conjugates comprising a (FKFE) 2 (SEQ ID NO: 1) beta- sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide, wherein the collagen hybridizing peptide comprises the sequence of at least (GXY)n (SEQ ID NO: 77), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is at least 3.
  • X can be proline, glutamic acid, or aspartic acid.
  • Y can be a modified proline, lysine, or arginine.
  • the modified proline can be hydroxy proline or fluoroproline.
  • a glycine can be modified as an aza-glycine.
  • a lysine can be modified as a pyrrolysine.
  • Collagen hybridizing peptides can comprise the sequence (Xaal-Xaa2-Xaa3)nl
  • Xaaa4- Xaa5-Xaa6 - (Xaa7-Xaa8-Xaa9)n2 (SEQ ID NOs:7-45), wherein Xaal, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, Xaa9 is glycine, proline, a modified proline or aza- glycine, and at least one of Xaal , Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, or Xaa9 is aza-glycine, wherein nl and n2 can be an integer from 1 to 20.
  • Collagen hybridizing peptides can comprise the sequence (Xaal-Xaa2-Xaa3)nl
  • Xaal, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, Xaa9 is glycine, proline, a modified proline or aza- glycine, and at least one of Xaal , Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, or Xaa9 is aza-glycine, wherein nl and n2 can be an integer from 1 to 20, wherein no more than one of Xaal , Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, or Xaa9 is aza
  • Xaal, Xaa2, and Xaa3 are not the same amino acid.
  • Xaa4, Xaa5, and Xaa6 are not the same amino acid.
  • Xaa7, Xaa8, and Xaa9 are not the same amino acid.
  • at least two of Xaal , Xaa2, and Xaa3 are not the same amino acid.
  • at least two of Xaa4, Xaa5, and Xaa6 are not the same amino acid.
  • at least two of Xaa7, Xaa8, and Xaa9 are not the same amino acid.
  • Collagen hybridizing peptides can comprise the sequence (Xaai-Xaa2-Xaa 3 ) n 1 - Xaa4- Xaa5-Xaa - (Xaa7-Xaa8-Xaaci)n 2 (SEQ ID NOs:7-45), wherein Xaai, Xaa2, Xaa3, Xaa4, Xaa 3 ⁇ 4 , Xaa , Xaa7, Xaag, Xaag is glycine, proline, a modified proline or aza-glycine, and at least one of Xaai, Xaa2, Xaa 3 , Xaa4, Xaa 3 ⁇ 4 , Xaa , Xaa7, Xaag, or Xaa 9 is aza-glycine, wherein the peptides comprises the sequence (Gly-Pro-Hyp) 3 -azGly
  • Collagen hybridizing peptides can the sequence (Xaai-Xaa2-Xaa 3 ) n 1 - Xaa4- Xaa5-Xaa - (Xaa7-Xaa8-Xaa9)n 2 (SEQ ID NOs:7-45), wherein Xaai, Xaa2, Xaa 3 , Xaa4, Xaa 3 ⁇ 4 , Xaa , Xaa7, Xaag, Xaag is glycine, proline, a modified proline or aza-glycine, and at least one of Xaai, Xaa2, Xaa 3 , Xaa4, Xaa 3 ⁇ 4 , Xaa , Xaa7, Xaag, or Xaag is aza-glycine, wherein n 1 and n 2 can be an integer from 1 to 20, wherein the peptides can have
  • Collagen hybridizing peptides can comprise the sequence (Xaai-Xaa2-Xaa 3 ) n 1 - Xaa4- Xaa5-Xaa - (Xaa7-Xaag-Xaa9)n 2 (SEQ ID NOs:7-45), wherein Xaai, Xaa2, Xaa 3 , Xaa4, Xaa 3 ⁇ 4 , Xa3 ⁇ 4, Xaa7, Xaag, Xaag is glycine, proline, a modified proline or aza-glycine, and at least one of Xaai, Xaa2, Xaa 3 , Xaa4, Xaa 3 ⁇ 4 , Xaa , Xaa7, Xaag, or Xaag is aza-glycine, wherein n 1 and n 2 can be an integer from 1 to 20, wherein the N-terminal amino
  • the collagen hybridizing peptides can comprise the sequence (Xi-Yi-Zi)-(X2-Y2-Z 2 )-(Xn-Yn-Zn) (SEQ ID NOs:63, 7-45), wherein n can be from 1-41, and X, Y, and Z are glycine, proline, a modified proline or aza-glycine, and at least one of the amino acids in the sequence is aza-glycine.
  • peptides that comprise between 1 and 41 amino acid triplets of the sequence X-Y-Z, wherein X, Y, and Z can be glycine, proline, a modified proline or aza-glycine and each triplet can have the same or different sequence.
  • the X, Y, and Z in each triplet can be the same as the X, Y, and Z in every triplet, respectively.
  • the X, Y, and Z in each triplet can be different from one or more of the X, Y, and Z's, respectively, in the other triplets.
  • collagen hybridizing peptides with the sequence (X 1 -Y 1 -Z 1 )- (X 2 -Y 2 -Z 2 )-(Xn-Yn-Zn) can include, but are not limited to AzGPO-GPO-AzGPO-GPO-GPO-GPO-GPO (SEQ ID NO:49); AzGPO-GPO-GPO- AzGPO-GPO-GPO-GPO (SEQ ID NO:50); GPO-AzGPO-AzGPO-GPO-GPO-GPO-GPO (SEQ ID NO:51); GPO-AzGPO-GPO-AzGPO-GPO-GPO-GPO (SEQ ID NO:52); GPO- GPO-AzGPO-GPO-AzGPO-GPO-GPO (SEQ ID NO:53); GPO-AzGPO-AzGPO- AzGPO-GPO-GPO-GPO-GPO (SEQ ID NO:53); GPO-Az
  • peptide conjugates comprising a (FKFE) 2 (SEQ ID NO: 1) beta- sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide, wherein the collagen hybridizing peptide is a dimeric collagen hybridizing peptide.
  • Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide; a linker; and a branch point, wherein the first and second collagen hybridizing peptides comprise the sequence of at least (GXY)n (SEQ ID NOs: 64-73), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is any number between 3 and 12
  • the first and second collagen hybridizing peptides are identical. In some instances, the first and second collagen hybridizing peptides are different. In some instances, the first and second collagen hybridizing peptides can be different in the sense that the sequences are different or they can have the same sequence but the number of repeats (i.e. n) is different.
  • Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide; a linker; and a branch point, wherein the first and second collagen hybridizing peptides comprise the sequence of at least (GXY)n (SEQ ID NOs: 64-73), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is any number between 3 and 12, wherein X is proline, glutamic acid, or aspartic acid.
  • Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide; a linker; and a branch point, wherein the first and second collagen hybridizing peptides comprise the sequence of at least (GXY)n (SEQ ID NOs: 64-73), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is any number between 3 and 12, wherein Y is a modified proline, lysine, or arginine.
  • G is glycine
  • X and Y are any amino acid
  • n is any number between 3 and 12
  • Y is a modified proline, lysine, or arginine.
  • X is proline, glutamic acid, or aspartic acid
  • Y is a modified proline, lysine, or arginine.
  • a modified proline can be hydroxyproline or fluoroproline.
  • Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide; a linker; and a branch point, wherein the first and second collagen hybridizing peptides comprise the sequence of at least (GXY)n (SEQ ID NOs: 64-73), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is any number between 3 and 12, wherein a glycine can be modified as an Aza-glycine. In some instances, only one glycine is modified as an Aza-glycine. In some instances, at least two glycines are modified as Aza-glycines. In some aspects, the X or Y can be Aza-glycines.
  • Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide; a linker; and a branch point, wherein the first and second collagen hybridizing peptides comprise the sequence of at least (GXY)n (SEQ ID NOs: 64-73), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is any number between 3 and 12, wherein the linker is between the collagen hybridizing peptides and the branch point. In some instances, there are at least two linkers. In some instances, the linker and branch point are on the C-terminal end of the first and second collagen hybridizing peptides.
  • the linker and branch point are on the N-terminal end of the first and second collagen hybridizing peptides.
  • the linker can be, but is not limited to, amino acid based or chemical.
  • the linker can be one or more glycine residues, aminohexanoic acid, or polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the linker can vary depending on whether the peptides are linked at the N-terminal end or the C-terminal end. For example, for N-terminal linking a two cysteine linker can be used and for C-terminal linking a reactive end linker to a template molecule such as diacid can be used.
  • Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide; a linker; and a branch point, wherein the first and second collagen hybridizing peptides comprise the sequence of at least (GXY)n (SEQ ID NOs: 64-73), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is any number between 3 and 12, wherein the branch point is a molecule that links the first and second collagen hybridizing peptides together through linkers attached to each first and second collagen hybridizing peptides.
  • the branch point can be amino acid based or a chemical compound.
  • the branch point can be a lysine residue.
  • the branch point can attach to a linker which is attached to the first collagen hybridizing peptide and to a linker which is attached to second collagen hybridizing peptide. Because the branch point attaches to a linker which attaches to the first and second collagen hybridizing peptides, the branch point is present on whichever end of the peptides the linker is located on. Thus, the branch point can be either on the N- terminal end or C-terminal end of the collagen hybridizing peptides.
  • Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide; a linker; and a branch point, wherein the first and second collagen hybridizing peptides comprise the sequence of at least (GXY)n (SEQ ID NOs: 64-73), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is any number between 3 and 12, wherein n can be 6 or 9.
  • peptide conjugates comprising an active agent; a spacer moiety; and a dimeric collagen hybridizing peptide comprising a first and second collagen hybridizing peptide; a linker; and a branch point
  • the first and second collagen hybridizing peptides comprise the sequence of at least (GXY)n (SEQ ID NOs:64-73), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is any number between 3 and 12, wherein the dimeric peptide can be represented by the formula [(Gly-Pro-Hyp)6-Gly-Gly-Gly]2-Lys, (Gly-Pro-Hyp)6-Gly-Gly-Gly-
  • the dimeric peptide comprises the formula [(Gly-Pro- Hyp) 9 -Gly-Gly-Gly] 2 -Lys, (Gly-Pro-Hyp) 9 -Gly-Gly-Gly - Lys - Gly-Gly-Gly - (Hyp- Pro
  • Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide; a linker; and a branch point, wherein the first and second collagen hybridizing peptides comprise the sequence of at least (GXY)n (SEQ ID NOs: 64-73), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is any number between 3 and 12, wherein the dimeric collagen hybridizing peptide can be attached to a solid support.
  • the solid support can be attached via an attachment point present between the branch point and the solid support.
  • the attachment point can be any amino acid residue.
  • the branch point also serves as the attachment point for the solid support.
  • the attachment point can be a glycine residue.
  • solid supports can be, but are not limited to, resin, polymeric beads, agarose beads, nanotubes, nanoparticles, surface coated with gold, acrylamide, cellulose, nitrocellulose, glass, gold, polystyrene, polyethylene vinyl acetate,
  • Solid supports can have any useful form including thin films or membranes, beads, bottles, dishes, fibers, optical fibers, woven fibers, chips, compact disks, shaped polymers, metals, particles and microparticles.
  • a chip is a rectangular or square small piece of material.
  • Dimeric collagen hybridizing peptides can also comprise a first and second collagen hybridizing peptide, a linker, and a branch point, wherein at least one of the first and second collagen hybridizing peptides comprises the sequence (Xaai-Xaa2-Xaa 3 )n 1 - Xaa4- Xaa 5 -Xaa - (Xaa7-Xaag-Xaaci)n 2 (SEQ ID NOs:7-45), wherein Xaai, Xaa2, Xaa 3 , Xaa4, Xaa5_ Xaa , Xaa7, Xaa8, Xaa 9 is glycine, proline, a modified proline or aza-glycine, and at least one of Xaai, Xaa2, Xaa 3 , Xaa4, Xaa 3 ⁇ 4 , Xa3 ⁇ 4, Xaa7, Xaag,
  • Xaai, Xaa2, Xaa3, Xaa4, Xaa3 ⁇ 4, Xaae, Xaa7, Xaag, or Xaag can be aza-glycine.
  • Xaai, Xaa2, and Xaa 3 are not the same amino acid.
  • Xaa4, Xaa 3 ⁇ 4 , and Xaa 3 ⁇ 4 are not the same amino acid.
  • Xaa7, Xaag, and Xaag are not the same amino acid.
  • At least two of Xaai, Xaa2, and Xaa 3 are not the same amino acid. In some instances, at least two of Xaa4, Xaa 3 ⁇ 4 , and Xaae are not the same amino acid. In some instances, at least two of Xaa7, Xaag, and Xaag are not the same amino acid.
  • Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide, a linker, and a branch point, wherein at least one of the first and second collagen hybridizing peptides comprises the sequence (Xaai-Xaa2-Xaa 3 )n 1 - Xaa4- Xaa5-Xaa - (Xaa7-Xaag-Xaaci)n 2 (SEQ ID NOs:7-45), wherein Xaai, Xaa2, Xaa 3 , Xaa4, Xaa5_ Xaa , Xaa7, Xaag, Xaag is glycine, proline, a modified proline or aza-glycine, and at least one of Xaai, Xaa2, Xaa 3 , Xaa4, Xaas , Xaa , Xaa7, Xaag, Xa
  • Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide, a linker, and a branch point, wherein at least one of the first and second collagen hybridizing peptides comprises the sequence (Xaai-Xaa2-Xaa 3 )n 1 - Xaa4- Xaa5-Xaae - (Xaa7-Xaag-Xaaci)n 2 (SEQ ID NOs:7-45), wherein Xaai, Xaa2, Xaa 3 , Xaa4, Xaa5_ Xaae, Xaa7, Xaa8, Xaag is glycine, proline, a modified proline or aza-glycine, and at least one of Xaai, Xaa2, Xaa 3 , Xaa4, Xaas , Xaae, Xaa7, Xaag, Xa
  • Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide, a linker, and a branch point, wherein at least one of the first and second collagen hybridizing peptides comprises the sequence (Xaai-Xaa2-Xaa 3 )n 1 - Xaa4- Xaa5-Xaae - (SEQ ID NOs:7-45), wherein Xaai, Xaa2, Xaa 3 , Xaa4, Xaa5_ Xaae, Xaa7, Xaa8, Xaag is glycine, proline, a modified proline or aza-glycine, and at least one of Xaai, Xaa2, Xaa 3 , Xaa4, Xaas , Xaae, Xaa7, Xaa8, Xaa 9 is aza-glycine, wherein the peptides have
  • Dimeric collagen hybridizing peptides can also comprise a first and second collagen hybridizing peptide, a linker, and a branch point, wherein at least one of the first and second collagen hybridizing peptides comprises the sequence (Xi-Yi_Zi)-(X2-Y2-Z2)- (Xn-Yn-Zn) (SEQ ID NO:63, 7-45), wherein n can be from 1 -41, and X, Y, and Z are glycine, proline, a modified proline or aza-glycine, and at least one of the amino acids in the sequence is aza-glycine.
  • peptides that comprise between 1 and 41 amino acid triplets of the sequence X-Y-Z, wherein X, Y, and Z can be glycine, proline, a modified proline or aza-glycine and each triplet can have the same or different sequence.
  • the X, Y, and Z in each triplet can be the same as the X, Y, and Z in every triplet, respectively.
  • the X, Y, and Z in each triplet can be different from one or more of the X, Y, and Z's, respectively, in the other triplets.
  • Dimeric collagen hybridizing peptides can also comprise a first and second collagen hybridizing peptide, a linker, and a branch point, wherein at least one of the first and second collagen hybridizing peptides comprises the sequence (Xi-Yi_Zi)-(X2-Y2-Z2)- (Xn-Yn-Zn) (SEQ ID NO:63, 7-45), wherein n can be from 1 -41, and X, Y, and Z are glycine, proline, a modified proline or aza-glycine, and at least one of the amino acids in the sequence is aza-glycine.
  • peptides with the sequence (X1-Y1-Z1XX2- Y 2 -Z 2 )-(Xn-Yn-Zn) can include, but are not limited to AzGPO- GPO-AzGPO-GPO-GPO-GPO-GPO (SEQ ID NO:49); AzGPO-GPO-GPO-AzGPO- GPO-GPO-GPO (SEQ ID NO:50); GPO-AzGPO-AzGPO-GPO-GPO-GPO-GPO-GPO (SEQ ID NO:51); GPO-AzGPO-GPO-AzGPO-GPO-GPO-GPO (SEQ ID NO:52); GPO-GPO- AzGPO-GPO-AzGPO-GPO-GPO (SEQ ID NO:53); GPO-AzGPO-AzGPO-AzGPO- GPO-GPO (SEQ ID NO:54);
  • compositions comprising one or more of the disclosed peptide conjugates.
  • compositions comprising one or more of the peptide conjugates comprising a (FKFE) 2 (SEQ ID NO: l) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
  • compositions can further comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material or carrier that would be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • carriers include dimyristoylphosphatidyl (DMPC), phosphate buffered saline or a multivesicular liposome.
  • PC Cholesterol: peptide or PC: peptide can be used as carriers in this invention.
  • Other suitable pharmaceutically acceptable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of
  • pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
  • the pH of the solution can be from about 5 to about 8 or from about 7 to about 7.5.
  • Further carriers include sustained release preparations such as semi-permeable matrices of solid hydrophobic polymers containing the composition, which matrices are in the form of shaped articles, e.g., films, stents (which are implanted in vessels during an angioplasty procedure), liposomes or microparticles.
  • compositions can also include carriers, thickeners, diluents, buffers, preservatives and the like, as long as the intended activity of the polypeptide, peptide, or conjugate of the invention is not compromised.
  • Pharmaceutical compositions may also include one or more active ingredients (in addition to the composition of the invention) such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.
  • nanofibers comprising two or more of the disclosed peptide conjugates.
  • nanofibers comprising two or more of the peptide conjugates comprising a (FKFE) 2 (SEQ ID NO: l) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
  • nanosheets comprising two or more of the disclosed nanofibers.
  • nanosheets comprising two or more of the nanofibers comprising two or more of the peptide conjugates comprising a (FKFE) 2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
  • FKFE FKFE 2
  • the two or more nanofibers are opposite orientations.
  • one nanofiber can have the collagen hybridizing peptides on the N-terminal end of the (FKFE) 2 (SEQ ID NO: 1) beta-sheet forming peptide and one nanofiber can have the collagen hybridizing peptides on the C-terminal end of the (FKFE) 2 (SEQ ID NO: 1) beta- sheet forming peptide.
  • detecting denatured collagen in a sample comprising contacting a composition comprising the one of the disclosed peptide conjugates, and detecting the presence or absence of binding of the peptide conjugate to denatured collagen, wherein the presence of binding indicates the presence of denatured collagen.
  • a peptide conjugate comprising a (FKFE) 2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide can be contacted with a sample.
  • detecting the presence or absence of binding of the peptide conjugate to denatured collagen can comprise an immunological assay.
  • immunological assays can include ELISAs.
  • peptide conjugates comprising a (FKFE) 2 (SEQ ID NO: l) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide can be administered.
  • Disclosed are methods of screening for a therapeutic agent that treats a disease or injury involving collagen damage comprising detecting the presence or absence of binding of a peptide conjugate to denatured collagen, the presence of binding indicating the presence of denatured collagen in the sample; administering to a sample having denatured collagen a composition comprising a peptide conjugates comprising a (FKFE) 2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide, wherein the active agent is a detectable moiety; and detecting the presence or absence of binding of the peptide conjugate to denatured collagen, the presence of binding indicating the presence of denatured collagen in the sample, wherein a decrease in the amount of damaged or denatured collagen in the sample detected after administration of the peptide conjugate compared to the amount detected prior to administration of the peptide conjugate indicates a therapeutic agent capable of a disease or injury involving collagen damage.
  • the detectable moiety comprising a
  • the disease or injury involving collagen damage can be cancer, osteoporosis, arthritis, fibrosis, a chronic wound, myocardial infarct,
  • Atherosclerotic plaques blood vessel injury and Failure (stroke), osteoarthritis, rheumatoid arthritis, osteoporosis, bone fracture/healing, Marfan syndrome, mechanical injury, sports medicine, e.g., rotator cuff tear, skin damage (photo, aging), skin cancer, skin
  • kits comprising one or more of the compositions described herein.
  • kits comprising a (FKFE)2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
  • kits comprising a peptide conjugate comprising a (FKFE)2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
  • kits can also include buffers, reagents, and instructions for how to make the peptide conjugates.
  • nanomaterials with controllable structural features.
  • the self-assembly properties of peptides have been exploited for generating many promising bio-inspired nanostructures, including nanofibers, nanotubes, and hydrogels.
  • the AFM height profiles of both NF-1 and NF-2 were estimated to 1.2 nm, indicating the formation of a double layer anti- -sheet by (FKFE) 2 (SEQ ID NO: 1). These results are consistent with previous reported results.
  • NF-1 and NF-2 are well dispersed and repel each other on their own, when mixed together, they showed strong tendency to further self-assemble into nanosheet (NS) ( Figure 1).
  • NS nanosheet
  • Figure 2E TEM examination of NS exhibited a well-defined assembly pattern, where NF-1 and NF-2 alternatively assembled
  • Figure 2F The AFM height profile of NS displayed an average value of approximately 1.2 nm ( Figure 2F, bottom panel), which is close to the AFM height values of both NF-1 and NF-2.
  • Peptide synthesis Peptides were synthesized on the TentaGel R RAM resin using standard F-moc chemistry using Focus XC peptide synthesizer (AAPPTec, Louisville, KY) with coupling cycles based on HBTU/DIEA-mediated (Adv Chemtech, Louisville, KY) activation. Five-fold molar excess of the amino acids and coupling reagents were used in a typical coupling reaction. The peptides were cleaved from the resins by treatment with water/l,2-ethanedithiol/thioanisole/trifluoroacetic acid
  • Nanofiber and nanosheet assembly Lyophilized peptide powder samples were dissolved in water to make 2 mM solutions, followed by heating these solutions up to 80°C for 5 min. These solutions were then cooled down to 4°C for at least 2 hours, to assemble into fibers.
  • NF-1 and NF-2 were mixed together to yield a final concentration of 1 mM of each in water. This mixture was then heated up to 60°C for 5 min, followed by cooling down to 4°C for at least 2 hours.
  • TEM Transmission electron microscopy
  • AFM Atomic force microscopy
  • CD spectra were recorded on a JASCO J-1500 CD (JASCO, Tokyo) in 0.10 mm quartz cells. All CHP samples were prepared in water and incubated at 4°C for at least 24 hr prior to CD measurement. Spectra were recorded from 190 to 300 nm at a scanning rate of 100 nm/min at 0.5 nm increment. 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. Melting temperatures were determined from the maximum of the first derivative of the melting curves.
  • Gelatin binding assay was performed by using an ELISA type plate. An ELISA type plate was prepared by coating with 0.5% gelatin solution at 4°C for 2 hours, with gentle shaking, then washed three times with PBS buffer, followed by blocking with 5% BSA at RT for 1 hour, washed three with PBS buffer prior to assay. Ten ⁇ of each fluorescent-labelled nanofiber was incubated with gelatin coated plate at 37°C for 2 hours, then washed three times with 37°C PBS buffer. SpectraMax M-2 microplate reader was used to measure the fluorescence (ex: 489 nm, em: 533 nm). Each binding experiment was done in triplicate.
  • FTIR Spectroscopy FTIR Spectroscopy measurement was acquired using a Nicolet iZIO Fourier transform infrared spectrometer (Thermo Scientific). Solution samples were freeze-dried into powder. The powder and potassium bromide were compressed into a thin pellet. Data were fitted using Gaussian function.
  • X-ray diffraction X-ray diffraction: Nanofibers/nanosheet solution samples were freeze- dried into powder, and loaded into capillary tubes. X-ray diffraction images were collected on a Rigaku Micromax-007 X-ray generator equipped with an R-Axis IV++ area detector.
  • Collagen hybridizing peptide-(FKFE) 2 (SEQ ID NO: 1) nanofibers and nanosheets can be used for staining tissue, ELISAs, tissue engineering, targeting and delivering systems, promoting T cell activity, and material sciences.
  • Staining tissue collagen hybridizing peptide-nanofibers can be used to amplify the signals, since they can carry large numbers of molecules in one fiber.
  • Developing ELISA type detection method The same type collagen hybridizing peptide-nanofibers are well dispersed and repel each other on their own, which can be applied to develop an ELISA type assay to detect diseased collagen fragments in body fluid.
  • an ELISA plate can firstly coat with collagen hybridizing peptide- nanofiber, followed by applying the sample on it, and finally detect with the same type collagen hybridizing peptide-nanofiber conjugated with signal molecules (ex.
  • Developing scaffold for tissue engineering collagen hybridizing peptide- nanofiber conjugated with therapeutic molecules can directly electron-spray with gelatin to make a gelatin-modified scaffold for would healing and tissue regeneration
  • Collagen hybridizing peptide-nanofiber can bind to denatured collagen, and most importantly, are very soluble and well-dispersed in buffer. Collagen hybridizing peptide-conjugated with therapeutic molecules can also target and deliver large numbers of therapeutic molecules to collagens undergoing degradation in vivo.
  • Promoting T cell activity When combined with collagen hybridizing peptides, the (FKFE) 2 (SEQ ID NO: l) nanofiber can become very inert, well-dispersed and highly soluble in buffer. Utilizing their dimensionally continuous structures, these nanofibers and nanosheets can be further engineered to tether large numbers of bio-macromolecules (such as, antibodies and proteins) to promote T cell activity. Nanosheets can also be used for clustering the T cell while stimulating them in T cell therapeutic applications.
  • peptides offer many advantages that allow us to control the formation of many defined structures (nanofibers, nanotubes, nanowires, etc), in addition to its modifiable ability, chemical stability, and inertness, as well as ease of large scale production.

Abstract

Disclosed are peptide conjugates comprising a (FKFE)2 (SEQ ID NO: l) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide. Disclosed are peptide conjugates comprising a (FKFE)2 (SEQ ID NO: l) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide, wherein the collagen hybridizing peptide comprises the sequence of at least (GXY)n (SEQ ID NO:77), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is at least 3. Disclosed are nanofibers comprising two or more of the disclosed peptide conjugates. Disclosed are nanosheets comprising two or more of the disclosed nanofibers. Disclosed are methods of detecting denatured collagen in a sample comprising contacting a composition comprising the one of the disclosed peptide conjugates, and detecting the presence or absence of binding of the peptide conjugate to denatured collagen, wherein the presence of binding indicates the presence of denatured collagen.

Description

COLLAGEN TARGETING NANOFIBERS AND NANOSHEETS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional Application No.
62/430,617, filed December 6, 2016, which is hereby incorporated herein by reference in its entirety.
REFERENCE TO SEQUENCE LISTING
[0002] The Sequence Listing submitted on February 27, 2017 as a text file named "21101_0337Pl_Sequence_Listing," created on February 22, 2017, and having a size of 70,377 bytes is hereby incorporated by reference.
BACKGROUND
[0003] Collagen hybridizing peptide (CHP) has an extremely strong tendency to self- assemble into a triple helix structure. However, in order to bind to denatured collagen, collagen hybridizing peptide needs to present in single strand or unfolded form. So far, strategies for either avoiding collagen hybridizing peptide self-trimerization or for collagen hybridizing peptides readily binding to denatured collagen are not well- developed, even though the use of these collagen hybridizing peptide-conjugated materials for many applications, including imaging, drug delivery, and tissue engineering can be crucial. The first strategy is to use heat treatment, since collagen hybridizing peptides are melted when they are heated above their melting temperatures. Recent studies indicated that, heat treatment can be applied to disrupt the trimeric form of collagen hybridizing peptides, which can target denatured collagen due to slow folding speed of the triple helix. The second strategy was recently introduced by inserting a photo-cleavable nitrobenzene group in the single strand collagen hybridizing peptide, caged collagen hybridizing peptide. This caged collagen hybridizing peptide triggers the triple peptides assembly by hindrance effect, and the assembly happens only upon shining a UV light on it. The former strategy using heat activated collagen hybridizing peptide seems to be ideal for collagen hybridizing peptide-based applications, especially in vivo therapeutics, because it eliminates potential toxicity of the photo-cleaved products and also simplifies the collagen hybridizing peptide structures for easy conjugation to other bioactive molecules. Even though both methods can generate single strand collagen hybridizing peptides, they still self-reassemble into triple helical structures when they are below their melting
temperatures, that prevents them bind to denatured collagen and thus significantly reduce their binding efficacy. Therefore, controlling collagen hybridizing peptide self-assembly while still retains its binding affinity to denatured collagen is highly desirable.
BRIEF SUMMARY
[0004] Disclosed are peptide conjugates comprising a (FKFE)2 (SEQ ID NO: 1) beta- sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
[0005] Disclosed are peptide conjugates comprising a (FKFE)2 (SEQ ID NO: 1) beta- sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide, wherein the collagen hybridizing peptide comprises the sequence of at least (GXY)n, wherein G is glycine, wherein X and Y are any amino acid, and wherein n is at least 3.
[0006] Disclosed are any of the disclosed peptide conjugates comprising a (FKFE)2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide, wherein the collagen hybridizing peptide can be on either the N-terminal end or C-terminal end of the (FKFE)2 (SEQ ID NO: l) beta-sheet forming peptide.
[0007] Disclosed are compositions comprising one or more of the disclosed peptide conjugates. For example, disclosed are compositions comprising one or more of the peptide conjugates comprising a (FKFE)2 (SEQ ID NO: l) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
[0008] Disclosed are nanofibers comprising two or more of the disclosed peptide conjugates. For example, disclosed are nanofibers comprising two or more of the peptide conjugates comprising a (FKFE)2 (SEQ ID NO: l) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
[0009] Disclosed are nanosheets comprising two or more of the disclosed nanofibers. For example, disclosed are nanosheets comprising two or more of the nanofibers comprising two or more of the peptide conjugates comprising a (FKFE)2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
[0010] Disclosed are methods of detecting denatured collagen in a sample comprising contacting a composition comprising the one of the disclosed peptide conjugates, and detecting the presence or absence of binding of the peptide conjugate to denatured collagen, wherein the presence of binding indicates the presence of denatured collagen. For example, a peptide conjugate comprising a (FKFE)2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide can be contacted with a sample.
[0011] Disclosed are methods of treating a disease or injury involving collagen damage comprising administering to a subject having a disease or injury involving collagen damage one of the disclosed peptide conjugates. For example, a peptide conjugate comprising a (FKFE)2 (SEQ ID NO: l) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide can be administered.
[0012] Disclosed are methods of screening for a therapeutic agent that treats a disease or injury involving collagen damage comprising detecting the presence or absence of binding of a peptide conjugate to denatured collagen, the presence of binding indicating the presence of denatured collagen in the sample; administering to a sample having denatured collagen a composition comprising a peptide conjugates comprising a (FKFE)2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide, wherein the active agent is a detectable moiety; and detecting the presence or absence of binding of the peptide conjugate to denatured collagen, the presence of binding indicating the presence of denatured collagen in the sample, wherein a decrease in the amount of damaged or denatured collagen in the sample detected after administration of the peptide conjugate compared to the amount detected prior to administration of the peptide conjugate indicates a therapeutic agent capable of a disease or injury involving collagen damage.
[0013] Disclosed are kits comprising a (FKFE)2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide. Also disclosed are kits comprising a peptide conjugate comprising a (FKFE)2 (SEQ ID NO: l) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
[0014] Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or may be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed method and
compositions and together with the description, serve to explain the principles of the disclosed method and compositions. [0016] Figure 1. Schematic of nanofibers and nanosheet structures. Nanofibers comprise of a CHP sequence of (GPO)9, (SEQ ID NO: 5), a linker G3, and an anti-parallel β-sheet forming peptide (FKFE)2 (SEQ ID NO: 1). Two types of CHP nanofiber with CHP of opposite polarity were combined with (FKFE)2 (SEQ ID NO: l): nanofiber with CHP oriented from N- to C-terminus, (GPO)9-G3-(FKFE)2 (SEQ ID NO:2) (designated as NF- 1), colored in blue; nanofiber with CHP oriented from C- to N-terminus, (FKFE)2-G3- (GPO)9 (SEQ ID NO:3) (designated as NF-2), colored in green. On their own, both nanofibers exhibited individual fiber in solution with no aggregation. When the two nanofibers are mixed together, they assemble into a single layer nanosheet (designated as NS) by triple helical folding of parallely-aligned CHPs. The blue and green arrows indicate the elongated direction of NF-1 and NF-2, respectively, as well as in the case of NS.
[0017] Figures 2A-2I. Transmission electron microscopy (TEM) and atomic force microscopy (AFM) images of nanofibers and nanosheet. (A) and (B) are TEM and AFM of NF-1, respectively. (D) and (E) are TEM and AFM of NF-2, respectively. (G) and (H) are TEM and AFM of NS, respectively. The blue arrows in each AFM indicate the width and the height where the measurements were taken. The bottom panel (C, F, I) in each AFM is the corresponding height and width profile with the number indicating the average width of the nanofibers.
[0018] Figures 3A-3B. Gelatin binding property of nanofibers. 5(6)- carboxy fluorescein (CF) conjugated nanofibers were used to study the binding to gelatin. (A) Ten μΜ of each nanofiber was incubated with gelatin coated ELISA-type plate at 37 °C for 2 hours. (B) Samples in (A) were washed at 37 °C overnight. NF-1 and NF-2 were pre-heated at 60 °C for 5 minutes to disrupt the potential intra-fiber CHP folding. (FKFE)2 (SEQ ID NO: 1) fiber alone and (FKFE)2 (SEQ ID NO: 1) fiber with a CHP scrambled sequence
Figure imgf000006_0001
(SEQ ID NO: 4) were used as controls.
[0019] Figure 4. Denatured collagen (Gelatin) behavior study of CHP-(FKFE)2 (SEQ ID NO: 1) nanofibers. The nanofibers were coated on a ELISA-type plate, followed by testing their binding behaviors with fluorescent-labelled CF-(GPO)9 (SEQ ID NO:5) and scrambled sequence CF-G9P9O9 (SEQ ID NO:6).
[0020] Figure 5. Specific binding of CHP -nanofiber.
[0021] Figure 6 is a schematic diagram of the basic structure of collagen is composed of a triple helix of 3 alpha chains. The most common amino acid motifs of collagen are glycine-proline-hydroxyproline, every third residue should be a glycine. The collagen mimetic peptide (CHP) is a synthetic peptide that mimics the basic structural motif of natural collagen— the triple helix, which shares the Gly-Pro-Hyp triplet. The Collagen Mimetic Peptide has a strong propensity to self-assemble into a triple helical structure.
[0022] Figure 7 is a schematic diagram of a single-strand synthetic CHP with a sequence from 6 to 10 (GPO) repeats that can specifically bind to denatured collagens through a unique triple-helix hybridization mechanism, which is similar to DNA hybridization.
[0023] Figure 8 is a schematic diagram showing that CHP has many potential applications, however, the problem with CHPs is that they have a high propensity to self- trimerize to form triple helical structure, and this will significantly reduce their targeting efficacy. The easy way to make CHP into single strand is to use heat to disrupt their triple helices. Another way to make CHP stay in single strand is to introduce a photo-cleavable nitrobenzene (NB) group in CHP to prevent them from self-trimerizing by hindrance effect, in this case a NB group is placed in the middle of CHP sequence. However, these strategies still cannot completely prevent them from self-trimerizing once they are cool down or the NB groups get removed by UV light. The disclosed CHP system is unable to self trimerize but still can target denatured collagen without any precondition like heating or UV -light irradiating.
[0024] Figure 9 is a schematic diagram showing how to turn CHP into nanofibers and nanosheets so that they can readily bind to denatured collagen with high affinity. CHP was linked to a short anti-parallel beta-sheet forming peptide, so that they can self- assemble into nanofibers and be able to bind to denatured collagen by multivalence effect.
[0025] Figures 10A, 10B, and IOC show the TEM and AFM images of the CHP-NF. No aggregation was seen. The dimensions of the NF are 1.5 nm in height, 22 nm in width, and over one micrometer in length.
[0026] Figure 11 shows circular dichroism (CD) measurement to investigate more about CHP-NF's secondary structure, it was confirmed that they assembled into a double- layered anti-parallel β-sheet nanofibers. The interesting thing is that a triple helical signal was not seen from CHP in NF.
[0027] Figure 12 shows the Tm measurement for this CHP-NF, unlike CHP peptide only which has a clear melting transition at about 75° C, no melting transition for CHP- NFs was seen, indicating that CHPs on NF did not self trimerize.
[0028] Figure 13 is a graph showing a gelatin behavior study of CHP-(FKFE)2 nanofibers. The nanofibers were coated on an ELISA-type plate, followed by testing their binding behaviours with fluorescent labeled CF-(GPO)9 (SEQ ID NO:5) and scrambled sequence CF-G9P9O9 (SEQ ID NO:6).
[0029] Figure 14 shows a longer CHP sequence can be displayed on the nanofiber platform and still can keep them for not self trimerizing. CHPs with sequences of (GPO) repeats up to 12 can be displayed. Again, no melting transitions were seen for CHPs in CD measurement, indicating that they did not form triple helical structures on the NFs. Normally, longer CHP sequences can give strong binding affinity, and the Tm of (GPO)i2 (SEQ ID NO: 76) is up to more than 100° C, which is very difficult to disrupt their triple helices by heating, also their refolding rate is very quick.
[0030] Figure 15 shows the binding affinity of CHP-NFs to denatured collagen using a denatured collagen binding assay. The ELISA type plate was first coated with denatured collagen or gelatin then fluorescently labeled CHP-NFs was applied on the wells at 37° C, for 2 h, followed by washing out the unbound NFs to check the remaining fluorescent signals. CHP-NFs showed exceptionally strong binding to denatured collagen at 37° C as compared with CHP alone, this can be due to multivalence effect.
[0031] Figure 16 shows that CHP-NFs were highly soluble in water and had no tendency to aggregate or bind to proteins other than denatured collagen due to the inertness and hydrophilicity of the CHP sequence. This is totally opposite with many other beta-sheet forming NFs, which normally tend to aggregate and form hydrogels.
[0032] Figures 17A-D shows that that CHP-NFs also behaved very similar to gelatin in cell culture, which means that they have no cytoxicity to cell culture.
[0033] Figure 18 shows that the length of the CHP-NFs can be controlled. The average length of the CHP-NFs is over 1 um. The NF length can be controlled by heating them in different temperatures. When we heated the NFs at 80° C/5 min, the NF length reduced to 200 nm, at 90° C/5 min, it further reduced to 100 -150 nm. No further change at longer heating time. No change in length was seen for 1 month incubation at 4° C, RT, or even at 37° C.
[0034] Figure 19 is a schematic diagram for testing the binding affinity of CHP-NFs in a mouse model. An IR dye was conjugated to the CHP-NF and injected into mice.
[0035] Figure 20 shows results in a mouse study. An IR680 dye was conjugated to the CHP-NF, and a NF was used with a CHP scramble sequence as a control. Without preheating the CHP-NF, they can readily bind to remodeling collagen in the bone. After one week, the signal is still very high. [0036] Figure 21 shows the CHP-nanosheet structure. To make CHP-nanosheets, 2 opposite polarity CHP-NFs were synthesized and then mixed together. Interestingly, these 2 different orientation NFs were able to form a sheet like structure, called the CHP- nanosheet. In order to form triple helical structures, CHPs need to be in the same orientation. Therefore, by displaying CHPs in opposite polarity on NFs, a CHP-nanosheet structure can be made, which is very unique.
[0037] Figures 22A-F show the TEM and AFM images of the CHP-nanosheet. 2 opposite polarity CHP nanofibers can form a monolayer CHP-nanosheet. (A) and (B) are TEM and AFM of NF-1, respectively. (C) and (D) are TEM and AFM of NF-2, respectively. (E) and (F) are TEM and AFM of NS, respectively.
[0038] Figure 23 shows the Tm for nanosheet. There is a clear melting transition for sample comprised NF-1 + NF-2. However, no melting transition was seen for the sample comprised of NF-1 + NF (NF with a CHP scramble sequence, used as a control), indicating that NF-1 and NF-2 are able to assemble into nanosheets via triple helical formation.
[0039] Figure 24 shows the TEM image of the nanosheet with NP on the surface, proving that the nanosheet can be easily functionalized with bioactive molecules, such as biotin.
[0040] Figure 25 shows the results of antibody binding to the nanosheet. The nanosheet was modified with even larger biomolecules, for example, in this case the nanosheet was functionalized with antibody, by adding an antibody binding motif to the nanosheet.
[0041] Figure 26 shows that this antibody functionalized NS is active. It was used to stimulate T cell activity. Because, it is well known that stimulating T cell using a stiff surface is more efficient than any other known methods. Here CD3 and CD28 antibodies were used to co-stimulate the T cells, and significant enhancement in cell activity was found as compared with the free antibodies, as evidenced by the IL-2 production. No significant difference was found between NF and NS.
[0042] Figure 27 shows that the nanosheet can cluster the T cells while stimulating the cells. T cell therapy has become one of the most promising treatments for many diseases such as cancers, but there are still many challenges (such as off target effect, delivering inactive cells, etc.) that need to be overcome before this method can be widely applied on patients. [0043] Figure 28 shows that in the control samples, no cell clusters were seen. Even though the NF can also promote the T cell activity it cannot cluster the cells.
[0044] Figure 29 shows that the nanosheet can also bind to denatured collagen while still binding to antibodies.
[0045] Figure 30 shows that T cells clustered by nanosheet can also bind to denatured collagen.
DETAILED DESCRIPTION
[0046] The disclosed method and compositions may be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.
[0047] It is to be understood that the disclosed method and compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
[0048] Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a collagen hybridizing peptide is disclosed and discussed and a number of modifications that can be made to a number of molecules are discussed, each and every combination and permutation of a collagen hybridizing peptide and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.
A. Definitions
[0049] It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
[0050] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide conjugate" includes a plurality of such peptide conjugates, reference to "the peptide conjugate" is a reference to one or more peptide conjugates and equivalents thereof known to those skilled in the art, and so forth.
[0051] "Optional" or "optionally" means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.
[0052] The term "treating" refers to partially or completely alleviating, ameliorating, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. For example, "treating" a disease or injury involving collagen damage can refer to reducing or eliminating the amount of damaged/denatured/diseased collagen. Treatment can also be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
[0053] The term "subject" refers to the target of administration, e.g. an animal. Thus the subject of the disclosed methods can be a vertebrate, such as a mammal. For example, the subject can be a human. The term does not denote a particular age or sex. Subject can be used interchangeably with "individual" or "patient."
[0054] Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range-1 from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. Finally, it should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.
[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.
[0056] Throughout the description and claims of this specification, the word
"comprise" and variations of the word, such as "comprising" and "comprises," means "including but not limited to," and is not intended to exclude, for example, other additives, components, integers or steps. In particular, in methods stated as comprising one or more steps or operations it is specifically contemplated that each step comprises what is listed (unless that step includes a limiting term such as "consisting of), meaning that each step is not intended to exclude, for example, other additives, components, integers or steps that are not listed in the step.
B. Peptide Conjugates
[0057] Disclosed are peptide conjugates comprising a (FKFE)2 (SEQ ID NO: 1) beta- sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
[0058] Also disclosed are peptide conjugates comprising a (FKFE)2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide, further comprising an active agent. In some instances, the active agent can be a detectable moiety or a therapeutic agent. A detectable moiety can be, but is not limited to, a fluorescent dye, radioactive isotope, magnetic bead, metallic bead, colloidal particle, near-infrared dye, or an electron-dense reagent. In some instances, the active agent can be attached to the N- terminal or C-terminal portion of the peptide conjugate. In some instances, the therapeutic agent can be a therapeutic known to treat a disease or injury involving collagen damage. For example, the therapeutic agent is an antibody, a peptide, a nucleic acid, or a chemical compound. In some instances, the disease or injury can be, but is not limited to, cancer, osteoporosis, arthritis, fibrosis, a chronic wound, myocardial infarct, atherosclerotic plaques, blood vessel injury and Failure (stroke), osteoarthritis, rheumatoid arthritis, osteoporosis, bone fracture/healing, Marfan syndrome, mechanical injury, sports medicine, e.g., rotator cuff tear, skin damage (photo, aging), skin cancer, skin inflammation, cornea injury & infection, corneal healing after LASIK, keratoconus, wet AMD, lung
inflammation, pulmonary fibrosis, liver fibrosis, cirrhosis, nephritis, kidney fibrosis, uterine fibroid, extracellular matrix (ECM) developmental biology, heritable connective tissue disorders, oral inflammation, cancer progression and metastasis, or foreign body response to implants.
1. Beta-sheet forming peptide
[0059] The disclosed (FKFE)2 (SEQ ID NO: 1) beta-sheet forming peptide comprises an alternating partem of hydrophobic and hydrophilic amino acids. (FKFE)2 (SEQ ID NO: 1) beta-sheet forming peptides can self-assemble into nanofibers. In some instances, the nanofibers can form nanosheets.
2. Spacer Moiety
[0060] Disclosed are any of the disclosed peptide conjugates comprising a (FKFE)2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide, wherein the spacer moiety is between the (FKFE)2 (SEQ ID NO: l) beta-sheet forming peptide and the collagen hybridizing peptide.
[0061] In some instances, the spacer moiety can comprise at least three glycines, aminohexanoic acid, gamma- Aminobutyric acid, (2-AMINOETHOXY)ACETIC ACID, 3-( amino)-3-(2-nitrophenyl)propionic acid or PEG.
[0062] In some instances, the spacer moiety can be any length and can be any molecule that helps link a (FKFE)2 (SEQ ID NO: 1) beta-sheet forming peptide and a collagen hybridizing peptide. In some instances, a spacer moiety can also be called a linker.
3. Collagen Hybridizing Peptides
[0063] Disclosed are peptide conjugates comprising a (FKFE)2 (SEQ ID NO: 1) beta- sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide, wherein the collagen hybridizing peptide comprises the sequence of at least (GXY)n (SEQ ID NO: 77), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is at least 3.
[0064] In some instances, X can be proline, glutamic acid, or aspartic acid. In some instances, Y can be a modified proline, lysine, or arginine. For example, the modified proline can be hydroxy proline or fluoroproline.
[0065] In some instances, a glycine can be modified as an aza-glycine.
[0066] In some instances, a lysine can be modified as a pyrrolysine.
[0067] Disclosed are any of the disclosed peptide conjugates comprising a (FKFE)2
(SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide, wherein the collagen hybridizing peptide can be on either the N-terminal end or
C-terminal end of the (FKFE)2 (SEQ ID NO: l) beta-sheet forming peptide.
i. Aza-Glycine Modified Collagen Hybridizing Peptides
[0068] Collagen hybridizing peptides can comprise the sequence (Xaal-Xaa2-Xaa3)nl
- Xaa4- Xaa5-Xaa6 - (Xaa7-Xaa8-Xaa9)n2 (SEQ ID NOs:7-45), wherein Xaal, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, Xaa9 is glycine, proline, a modified proline or aza- glycine, and at least one of Xaal , Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, or Xaa9 is aza-glycine, wherein nl and n2 can be an integer from 1 to 20.
[0069] Collagen hybridizing peptides can comprise the sequence (Xaal-Xaa2-Xaa3)nl
- Xaa4- Xaa5-Xaa6 - (Xaa7-Xaa8-Xaa9)n2 (SEQ ID NOs:7-45), wherein Xaal, Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, Xaa9 is glycine, proline, a modified proline or aza- glycine, and at least one of Xaal , Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, or Xaa9 is aza-glycine, wherein nl and n2 can be an integer from 1 to 20, wherein no more than one of Xaal , Xaa2, Xaa3, Xaa4, Xaa5, Xaa6, Xaa7, Xaa8, or Xaa9 is aza-glycine. In some instances, Xaal, Xaa2, and Xaa3 are not the same amino acid. In some instances, Xaa4, Xaa5, and Xaa6 are not the same amino acid. In some instances, Xaa7, Xaa8, and Xaa9 are not the same amino acid. In some instances, at least two of Xaal , Xaa2, and Xaa3 are not the same amino acid. In some instances, at least two of Xaa4, Xaa5, and Xaa6 are not the same amino acid. In some instances, at least two of Xaa7, Xaa8, and Xaa9 are not the same amino acid.
[0070] Collagen hybridizing peptides can comprise the sequence (Xaai-Xaa2-Xaa3)n 1 - Xaa4- Xaa5-Xaa - (Xaa7-Xaa8-Xaaci)n2 (SEQ ID NOs:7-45), wherein Xaai, Xaa2, Xaa3, Xaa4, Xaa¾, Xaa , Xaa7, Xaag, Xaag is glycine, proline, a modified proline or aza-glycine, and at least one of Xaai, Xaa2, Xaa3, Xaa4, Xaa¾, Xaa , Xaa7, Xaag, or Xaa9 is aza-glycine, wherein the peptides comprises the sequence (Gly-Pro-Hyp)3-azGly-Pro-Hyp-(Gly -Pro- Hyp^ (SEQ ID NO:46); (Pro-Hyp-Gly)3-Pro-Hyp-azGly- (Pro-Hyp-Gly)3 (SEQ ID NO:47); or (Pro-Hyp-Gly)3-Pro-Pro-azGly-(Pro-Hyp-Gly)3 (SEQ ID NO:48).
[0071] Collagen hybridizing peptides can the sequence (Xaai-Xaa2-Xaa3)n 1 - Xaa4- Xaa5-Xaa - (Xaa7-Xaa8-Xaa9)n2 (SEQ ID NOs:7-45), wherein Xaai, Xaa2, Xaa3, Xaa4, Xaa¾, Xaa , Xaa7, Xaag, Xaag is glycine, proline, a modified proline or aza-glycine, and at least one of Xaai, Xaa2, Xaa3, Xaa4, Xaa¾, Xaa , Xaa7, Xaag, or Xaag is aza-glycine, wherein n1 and n2 can be an integer from 1 to 20, wherein the peptides can have a higher affinity to degraded collagen than a conventional collagen hybridizing peptide. In some instances, the peptides do not bind native collagen.
[0072] Collagen hybridizing peptides can comprise the sequence (Xaai-Xaa2-Xaa3)n 1 - Xaa4- Xaa5-Xaa - (Xaa7-Xaag-Xaa9)n2 (SEQ ID NOs:7-45), wherein Xaai, Xaa2, Xaa3, Xaa4, Xaa¾, Xa¾, Xaa7, Xaag, Xaag is glycine, proline, a modified proline or aza-glycine, and at least one of Xaai, Xaa2, Xaa3, Xaa4, Xaa¾, Xaa , Xaa7, Xaag, or Xaag is aza-glycine, wherein n1 and n2 can be an integer from 1 to 20, wherein the N-terminal amino acid comprises an acetyl group. In some instances, the C-terminal amino acid comprises an amino group.
[0073] In some instances, the collagen hybridizing peptides can comprise the sequence (Xi-Yi-Zi)-(X2-Y2-Z2)-(Xn-Yn-Zn) (SEQ ID NOs:63, 7-45), wherein n can be from 1-41, and X, Y, and Z are glycine, proline, a modified proline or aza-glycine, and at least one of the amino acids in the sequence is aza-glycine. Disclosed are peptides that comprise between 1 and 41 amino acid triplets of the sequence X-Y-Z, wherein X, Y, and Z can be glycine, proline, a modified proline or aza-glycine and each triplet can have the same or different sequence. In some instances, the X, Y, and Z in each triplet can be the same as the X, Y, and Z in every triplet, respectively. In some instances, the X, Y, and Z in each triplet can be different from one or more of the X, Y, and Z's, respectively, in the other triplets.
[0074] In some instances, collagen hybridizing peptides with the sequence (X1-Y1-Z1)- (X2-Y2-Z2)-(Xn-Yn-Zn) (SEQ ID NOs:63, 7-45) can include, but are not limited to AzGPO-GPO-AzGPO-GPO-GPO-GPO-GPO (SEQ ID NO:49); AzGPO-GPO-GPO- AzGPO-GPO-GPO-GPO (SEQ ID NO:50); GPO-AzGPO-AzGPO-GPO-GPO-GPO-GPO (SEQ ID NO:51); GPO-AzGPO-GPO-AzGPO-GPO-GPO-GPO (SEQ ID NO:52); GPO- GPO-AzGPO-GPO-AzGPO-GPO-GPO (SEQ ID NO:53); GPO-AzGPO-AzGPO- AzGPO-GPO-GPO-GPO (SEQ ID NO:54); AzGPO-GPO-AzGPO-AzGPO-GPO-GPO- GPO (SEQ ID NO:55); AzGPO-GPO-AzGPO-GPO-AzGPO-GPO-GPO (SEQ ID
NO:56); AzGPO-GPO-AzGPO-GPO-GPO-AzGPO-GPO (SEQ ID NO:57); GPO- AzGPO-GPO-AzGPO-GPO-AzGPO-GPO (SEQ ID NO:58); AzGPO-GPO-AzGPO- AzGPO-AzGPO-GPO-GPO (SEQ ID NO:59); AzGPO-GPO-GPO-AzGPO-AzGPO- AzGPO-GPO (SEQ ID NO:60); AzGPO-GPO-AzGPO-GPO-AzGPO-AzGPO-GPO (SEQ ID NO:61); AzGPO-GPO-AzGPO-GPO-AzGPO-GPO-AzGPO (SEQ ID NO:62).
ii. Dimeric Collagen Hybridizing Peptides
[0075] Disclosed are peptide conjugates comprising a (FKFE)2 (SEQ ID NO: 1) beta- sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide, wherein the collagen hybridizing peptide is a dimeric collagen hybridizing peptide.
[0076] Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide; a linker; and a branch point, wherein the first and second collagen hybridizing peptides comprise the sequence of at least (GXY)n (SEQ ID NOs: 64-73), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is any number between 3 and 12
[0077] In some instances, the first and second collagen hybridizing peptides are identical. In some instances, the first and second collagen hybridizing peptides are different. In some instances, the first and second collagen hybridizing peptides can be different in the sense that the sequences are different or they can have the same sequence but the number of repeats (i.e. n) is different.
[0078] Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide; a linker; and a branch point, wherein the first and second collagen hybridizing peptides comprise the sequence of at least (GXY)n (SEQ ID NOs: 64-73), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is any number between 3 and 12, wherein X is proline, glutamic acid, or aspartic acid.
[0079] Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide; a linker; and a branch point, wherein the first and second collagen hybridizing peptides comprise the sequence of at least (GXY)n (SEQ ID NOs: 64-73), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is any number between 3 and 12, wherein Y is a modified proline, lysine, or arginine. In some instances, X is proline, glutamic acid, or aspartic acid and Y is a modified proline, lysine, or arginine. A modified proline can be hydroxyproline or fluoroproline.
[0080] Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide; a linker; and a branch point, wherein the first and second collagen hybridizing peptides comprise the sequence of at least (GXY)n (SEQ ID NOs: 64-73), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is any number between 3 and 12, wherein a glycine can be modified as an Aza-glycine. In some instances, only one glycine is modified as an Aza-glycine. In some instances, at least two glycines are modified as Aza-glycines. In some aspects, the X or Y can be Aza-glycines.
[0081] Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide; a linker; and a branch point, wherein the first and second collagen hybridizing peptides comprise the sequence of at least (GXY)n (SEQ ID NOs: 64-73), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is any number between 3 and 12, wherein the linker is between the collagen hybridizing peptides and the branch point. In some instances, there are at least two linkers. In some instances, the linker and branch point are on the C-terminal end of the first and second collagen hybridizing peptides. In some instances, the linker and branch point are on the N-terminal end of the first and second collagen hybridizing peptides. In some instances, the linker can be, but is not limited to, amino acid based or chemical. For example, the linker can be one or more glycine residues, aminohexanoic acid, or polyethylene glycol (PEG). The linker can vary depending on whether the peptides are linked at the N-terminal end or the C-terminal end. For example, for N-terminal linking a two cysteine linker can be used and for C-terminal linking a reactive end linker to a template molecule such as diacid can be used.
[0082] Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide; a linker; and a branch point, wherein the first and second collagen hybridizing peptides comprise the sequence of at least (GXY)n (SEQ ID NOs: 64-73), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is any number between 3 and 12, wherein the branch point is a molecule that links the first and second collagen hybridizing peptides together through linkers attached to each first and second collagen hybridizing peptides. The branch point can be amino acid based or a chemical compound. For example, in some instances, the branch point can be a lysine residue. In some instances, the branch point can attach to a linker which is attached to the first collagen hybridizing peptide and to a linker which is attached to second collagen hybridizing peptide. Because the branch point attaches to a linker which attaches to the first and second collagen hybridizing peptides, the branch point is present on whichever end of the peptides the linker is located on. Thus, the branch point can be either on the N- terminal end or C-terminal end of the collagen hybridizing peptides.
[0083] Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide; a linker; and a branch point, wherein the first and second collagen hybridizing peptides comprise the sequence of at least (GXY)n (SEQ ID NOs: 64-73), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is any number between 3 and 12, wherein n can be 6 or 9. Disclosed are peptide conjugates comprising an active agent; a spacer moiety; and a dimeric collagen hybridizing peptide comprising a first and second collagen hybridizing peptide; a linker; and a branch point, wherein the first and second collagen hybridizing peptides comprise the sequence of at least (GXY)n (SEQ ID NOs:64-73), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is any number between 3 and 12, wherein the dimeric peptide can be represented by the formula [(Gly-Pro-Hyp)6-Gly-Gly-Gly]2-Lys, (Gly-Pro-Hyp)6-Gly-Gly-
(^Y-Pro- I^Bjs-ilSy-Gly-GIy— Lys
Gly - Lys - Gly-Gly-Gly - (Hyp- Pro -Gly)6, or iGly-Pro-H^-GIy-Gfy-Gly SEQ ID NO:74) . In some instances, the dimeric peptide comprises the formula [(Gly-Pro- Hyp)9-Gly-Gly-Gly]2-Lys, (Gly-Pro-Hyp)9-Gly-Gly-Gly - Lys - Gly-Gly-Gly - (Hyp- Pro
Figure imgf000018_0001
-Gly)9, or » FEo-SSR -Gl -Giy-GIy (SEQ m NO:75).
[0084] Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide; a linker; and a branch point, wherein the first and second collagen hybridizing peptides comprise the sequence of at least (GXY)n (SEQ ID NOs: 64-73), wherein G is glycine, wherein X and Y are any amino acid, and wherein n is any number between 3 and 12, wherein the dimeric collagen hybridizing peptide can be attached to a solid support. In some instances, the solid support can be attached via an attachment point present between the branch point and the solid support. In some instances, the attachment point can be any amino acid residue. In some instances, the branch point also serves as the attachment point for the solid support. For example, the attachment point can be a glycine residue. In some instances, solid supports can be, but are not limited to, resin, polymeric beads, agarose beads, nanotubes, nanoparticles, surface coated with gold, acrylamide, cellulose, nitrocellulose, glass, gold, polystyrene, polyethylene vinyl acetate,
polypropylene, polymethacrylate, polyethylene, polyethylene oxide, glass, polysilicates, polycarbonates, teflon, fluorocarbons, nylon, silicon rubber, polyanhydrides, polygly colic acid, polylactic acid, polyorthoesters, functionalized silane, polypropylfumerate, collagen, glycosaminoglycans, and polyamino acids or any polymeric surface. Solid supports can have any useful form including thin films or membranes, beads, bottles, dishes, fibers, optical fibers, woven fibers, chips, compact disks, shaped polymers, metals, particles and microparticles. A chip is a rectangular or square small piece of material.
[0085] Dimeric collagen hybridizing peptides can also comprise a first and second collagen hybridizing peptide, a linker, and a branch point, wherein at least one of the first and second collagen hybridizing peptides comprises the sequence (Xaai-Xaa2-Xaa3)n1 - Xaa4- Xaa5-Xaa - (Xaa7-Xaag-Xaaci)n2 (SEQ ID NOs:7-45), wherein Xaai, Xaa2, Xaa3, Xaa4, Xaa5_ Xaa , Xaa7, Xaa8, Xaa9 is glycine, proline, a modified proline or aza-glycine, and at least one of Xaai, Xaa2, Xaa3, Xaa4, Xaa¾, Xa¾, Xaa7, Xaag, or Xaa9 is aza-glycine. In some instances, wherein no more than one of Xaai, Xaa2, Xaa3, Xaa4, Xaa¾, Xaae, Xaa7, Xaag, or Xaagcan be aza-glycine. In some instances, Xaai, Xaa2, and Xaa3 are not the same amino acid. In some instances, Xaa4, Xaa¾, and Xaa¾ are not the same amino acid. In some instances, Xaa7, Xaag, and Xaag are not the same amino acid. In some instances, at least two of Xaai, Xaa2, and Xaa3 are not the same amino acid. In some instances, at least two of Xaa4, Xaa¾, and Xaae are not the same amino acid. In some instances, at least two of Xaa7, Xaag, and Xaag are not the same amino acid.
[0086] Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide, a linker, and a branch point, wherein at least one of the first and second collagen hybridizing peptides comprises the sequence (Xaai-Xaa2-Xaa3)n1 - Xaa4- Xaa5-Xaa - (Xaa7-Xaag-Xaaci)n2 (SEQ ID NOs:7-45), wherein Xaai, Xaa2, Xaa3, Xaa4, Xaa5_ Xaa , Xaa7, Xaag, Xaag is glycine, proline, a modified proline or aza-glycine, and at least one of Xaai, Xaa2, Xaa3, Xaa4, Xaas, Xaa , Xaa7, Xaag, Xaag is aza-glycine, wherein the peptides comprise the sequence (Gly-Pro-Hyp)3-azGly-Pro-Hyp-(Gly-Pro-Hyp)3 (SEQ ID NO:46), (Pro-Hyp-Gly)3-Pro-Hyp-azGly- (Pro-Hyp-Gly)3 (SEQ ID NO:47), or (Pro- Hyp-Gly)3-Pro-Pro-azGly-(Pro-Hyp-Gly)3 (SEQ ID NO:48).
[0087] Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide, a linker, and a branch point, wherein at least one of the first and second collagen hybridizing peptides comprises the sequence (Xaai-Xaa2-Xaa3)n1 - Xaa4- Xaa5-Xaae - (Xaa7-Xaag-Xaaci)n2 (SEQ ID NOs:7-45), wherein Xaai, Xaa2, Xaa3, Xaa4, Xaa5_ Xaae, Xaa7, Xaa8, Xaag is glycine, proline, a modified proline or aza-glycine, and at least one of Xaai, Xaa2, Xaa3, Xaa4, Xaas, Xaae, Xaa7, Xaag, Xaag is aza-glycine, wherein n1 can be an integer from 1 to 20. In some instances, n2 can be an integer from 1 to 20.
[0088] Dimeric collagen hybridizing peptides can comprise a first and second collagen hybridizing peptide, a linker, and a branch point, wherein at least one of the first and second collagen hybridizing peptides comprises the sequence (Xaai-Xaa2-Xaa3)n1 - Xaa4- Xaa5-Xaae -
Figure imgf000020_0001
(SEQ ID NOs:7-45), wherein Xaai, Xaa2, Xaa3, Xaa4, Xaa5_ Xaae, Xaa7, Xaa8, Xaag is glycine, proline, a modified proline or aza-glycine, and at least one of Xaai, Xaa2, Xaa3, Xaa4, Xaas, Xaae, Xaa7, Xaa8, Xaa9 is aza-glycine, wherein the peptides have a higher affinity to degraded collagen than a conventional collagen hybridizing peptide. In some instances, the peptides do not bind native collagen.
[0089] Dimeric collagen hybridizing peptides can also comprise a first and second collagen hybridizing peptide, a linker, and a branch point, wherein at least one of the first and second collagen hybridizing peptides comprises the sequence (Xi-Yi_Zi)-(X2-Y2-Z2)- (Xn-Yn-Zn) (SEQ ID NO:63, 7-45), wherein n can be from 1 -41, and X, Y, and Z are glycine, proline, a modified proline or aza-glycine, and at least one of the amino acids in the sequence is aza-glycine. Disclosed are peptides that comprise between 1 and 41 amino acid triplets of the sequence X-Y-Z, wherein X, Y, and Z can be glycine, proline, a modified proline or aza-glycine and each triplet can have the same or different sequence. In some instances, the X, Y, and Z in each triplet can be the same as the X, Y, and Z in every triplet, respectively. In some instances, the X, Y, and Z in each triplet can be different from one or more of the X, Y, and Z's, respectively, in the other triplets.
[0090] Dimeric collagen hybridizing peptides can also comprise a first and second collagen hybridizing peptide, a linker, and a branch point, wherein at least one of the first and second collagen hybridizing peptides comprises the sequence (Xi-Yi_Zi)-(X2-Y2-Z2)- (Xn-Yn-Zn) (SEQ ID NO:63, 7-45), wherein n can be from 1 -41, and X, Y, and Z are glycine, proline, a modified proline or aza-glycine, and at least one of the amino acids in the sequence is aza-glycine. In some instances, peptides with the sequence (X1-Y1-Z1XX2- Y2-Z2)-(Xn-Yn-Zn) (SEQ ID NOs:63, 7-45) can include, but are not limited to AzGPO- GPO-AzGPO-GPO-GPO-GPO-GPO (SEQ ID NO:49); AzGPO-GPO-GPO-AzGPO- GPO-GPO-GPO (SEQ ID NO:50); GPO-AzGPO-AzGPO-GPO-GPO-GPO-GPO (SEQ ID NO:51); GPO-AzGPO-GPO-AzGPO-GPO-GPO-GPO (SEQ ID NO:52); GPO-GPO- AzGPO-GPO-AzGPO-GPO-GPO (SEQ ID NO:53); GPO-AzGPO-AzGPO-AzGPO- GPO-GPO-GPO (SEQ ID NO:54); AzGPO-GPO-AzGPO-AzGPO-GPO-GPO-GPO (SEQ ID NO:55); AzGPO-GPO-AzGPO-GPO-AzGPO-GPO-GPO (SEQ ID NO:56); AzGPO- GPO-AzGPO-GPO-GPO-AzGPO-GPO (SEQ ID NO:57); GPO-AzGPO-GPO-AzGPO- GPO-AzGPO-GPO (SEQ ID NO:58); AzGPO-GPO-AzGPO-AzGPO-AzGPO-GPO-GPO (SEQ ID NO:59); AzGPO-GPO-GPO-AzGPO-AzGPO-AzGPO-GPO (SEQ ID NO:60); AzGPO-GPO-AzGPO-GPO-AzGPO-AzGPO-GPO (SEQ ID NO:61); AzGPO-GPO- AzGPO-GPO-AzGPO-GPO-AzGPO (SEQ ID NO:62).
C. Compositions
[0091] Disclosed are compositions comprising one or more of the disclosed peptide conjugates. For example, disclosed are compositions comprising one or more of the peptide conjugates comprising a (FKFE)2 (SEQ ID NO: l) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
[0092] In some instances, the disclosed compositions can further comprise a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material or carrier that would be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art. Examples of carriers include dimyristoylphosphatidyl (DMPC), phosphate buffered saline or a multivesicular liposome. For example,
PG: PC: Cholesterol: peptide or PC: peptide can be used as carriers in this invention. Other suitable pharmaceutically acceptable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, an appropriate amount of
pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Other examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution can be from about 5 to about 8 or from about 7 to about 7.5. Further carriers include sustained release preparations such as semi-permeable matrices of solid hydrophobic polymers containing the composition, which matrices are in the form of shaped articles, e.g., films, stents (which are implanted in vessels during an angioplasty procedure), liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH.
[0093] Pharmaceutical compositions can also include carriers, thickeners, diluents, buffers, preservatives and the like, as long as the intended activity of the polypeptide, peptide, or conjugate of the invention is not compromised. Pharmaceutical compositions may also include one or more active ingredients (in addition to the composition of the invention) such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.
D. Nanofibers and Nanosheets
[0094] Disclosed are nanofibers comprising two or more of the disclosed peptide conjugates. For example, disclosed are nanofibers comprising two or more of the peptide conjugates comprising a (FKFE)2 (SEQ ID NO: l) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
[0095] Disclosed are nanosheets comprising two or more of the disclosed nanofibers. For example, disclosed are nanosheets comprising two or more of the nanofibers comprising two or more of the peptide conjugates comprising a (FKFE)2(SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
[0096] In some instances, the two or more nanofibers are opposite orientations. For example, one nanofiber can have the collagen hybridizing peptides on the N-terminal end of the (FKFE)2 (SEQ ID NO: 1) beta-sheet forming peptide and one nanofiber can have the collagen hybridizing peptides on the C-terminal end of the (FKFE)2 (SEQ ID NO: 1) beta- sheet forming peptide.
E. Methods
[0097] Disclosed are methods of detecting denatured collagen in a sample comprising contacting a composition comprising the one of the disclosed peptide conjugates, and detecting the presence or absence of binding of the peptide conjugate to denatured collagen, wherein the presence of binding indicates the presence of denatured collagen. For example, a peptide conjugate comprising a (FKFE)2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide can be contacted with a sample. [0098] In some instances, detecting the presence or absence of binding of the peptide conjugate to denatured collagen can comprise an immunological assay. For example, immunological assays can include ELISAs.
[0099] Disclosed are methods of treating a disease or injury involving collagen damage comprising administering to a subject having a disease or injury involving collagen damage one of the disclosed peptide conjugates. For example, a peptide conjugate comprising a (FKFE)2 (SEQ ID NO: l) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide can be administered.
[00100] Disclosed are methods of screening for a therapeutic agent that treats a disease or injury involving collagen damage comprising detecting the presence or absence of binding of a peptide conjugate to denatured collagen, the presence of binding indicating the presence of denatured collagen in the sample; administering to a sample having denatured collagen a composition comprising a peptide conjugates comprising a (FKFE)2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide, wherein the active agent is a detectable moiety; and detecting the presence or absence of binding of the peptide conjugate to denatured collagen, the presence of binding indicating the presence of denatured collagen in the sample, wherein a decrease in the amount of damaged or denatured collagen in the sample detected after administration of the peptide conjugate compared to the amount detected prior to administration of the peptide conjugate indicates a therapeutic agent capable of a disease or injury involving collagen damage. In some instances, the detectable moiety can be, but is not limited to, a fluorescent dye, radioactive isotope, magnetic bead, metallic bead, colloidal particle, near- infrared dye, or an electron-dense reagent.
[00101] In some instances, the disease or injury involving collagen damage can be cancer, osteoporosis, arthritis, fibrosis, a chronic wound, myocardial infarct,
atherosclerotic plaques, blood vessel injury and Failure (stroke), osteoarthritis, rheumatoid arthritis, osteoporosis, bone fracture/healing, Marfan syndrome, mechanical injury, sports medicine, e.g., rotator cuff tear, skin damage (photo, aging), skin cancer, skin
inflammation, cornea injury & infection, corneal healing after LASIK, keratoconus, wet AMD, lung inflammation, pulmonary fibrosis, liver fibrosis, cirrhosis, nephritis, kidney fibrosis, uterine fibroid, extracellular matrix (ECM) developmental biology, heritable connective tissue disorders, oral inflammation, cancer progression and metastasis, or foreign body response to implants. F. Kits
[00102] The materials described above as well as other materials can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the disclosed method. It is useful if the kit components in a given kit are designed and adapted for use together in the disclosed method. Disclosed herein are kits comprising one or more of the compositions described herein. For example disclosed are kits comprising a (FKFE)2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide. Also disclosed are kits comprising a peptide conjugate comprising a (FKFE)2 (SEQ ID NO: 1) beta-sheet forming peptide; a spacer moiety; and a collagen hybridizing peptide.
[00103] The disclosed kits can also include buffers, reagents, and instructions for how to make the peptide conjugates.
Examples
A. Self-assembled collagen mimetic peptide nanofibers and nanosheet
[00104] Self-assembled peptide nanostructures have demonstrated as promising biomaterials for various potential applications such as tissue engineering, drug delivery, wound healing, and vaccines. The versatility of the peptides, together with their ability to form specific secondary structures, offers a unique platform for the design of
nanomaterials with controllable structural features. The self-assembly properties of peptides have been exploited for generating many promising bio-inspired nanostructures, including nanofibers, nanotubes, and hydrogels.
[00105] To explore and maximize the collagen hybridizing pepti de-mediated targeting capacity for imaging and for delivering therapeutic drugs to collagens undergo degradation, we developed a self-assembled peptide nanofiber that can tether a large amount of single strand collagen hybridizing peptides, and the most importantly, they can readily bind to denatured collagen. Utilizing the unique property of self-assembly peptides, we combined our collagen hybridizing peptides with a β-sheet forming peptide to make a collagen hybridizing peptide-nanofiber. A short peptide (FKFEFKFE) or (FKFE)2 (SEQ ID NO: l), comprising of an alternating pattern of hydrophobic and hydrophilic amino acids, was chosen to combine with collagen hybridizing peptides (collagen hybridizing peptide-(FKFE)2 (SEQ ID NO: 1). Since it is known that peptides containing alternating hydrophilic and hydrophobic amino acid residues have a strong tendency to generate β-sheet structures. This (FKFE)2 (SEQ ID NO: l) peptide was shown to form self- assembled nanofiber structures. It initially formed helical ribbon, then converted into nanofiber structures, and favorably transformed into large aggregated nanostructures. Molecular dynamics simulation study of this nanofiber suggested the double helical anti-β- sheet favorable formation (refs). Similarly, we expected that collagen hybridizing peptide- (FKFE)2 (SEQ ID NO: l) peptides can also assemble into nanofibers with collagen hybridizing peptides exposed outside (Figure 1). Additionally, we hypothesized that the triple helical folding between collagen hybridizing peptides of correct geometry displayed on collagen hybridizing peptide-(FKFE)2 (SEQ ID NO: l) fibers can further assemble into nanosheet.
[00106] To investigate the behavior of collagen hybridizing peptide-(FKFE)2 (SEQ ID NO: l) assembly resulting from the geometry of collagen hybridizing peptide display, we synthesized (FKFE)2 (SEQ ID NO: 1) having collagen hybridizing peptides at either N terminus (GPO)9-G3-(FKFE)2 (SEQ ID NO:2) (designated as NF-1), or C terminus (FKFE)2-G3-(GPO)9 (SEQ ID NO:3) (designated as NF-2) (Figure 1). Triple glycine was used as a linker. Transmission electron microscopy (TEM) analysis showed that both NF- 1 and NF-2 peptides self-assembled into well-dispersed nanofibers (Figure 2). They both have a central core of (FKFE)2 (SEQ ID NO: 1) structure with the collagen hybridizing peptides exposed outside. The width of the (FKFE)2 (SEQ ID NO: 1) central core was measured to be approximately 7 nm by TEM. The overall width of NF-1 and NF-2 were further confirmed by atomic force microscopy (AFM) measurements, the results revealed that NF-1 and NF-2 had widths of about 30 and 20 nm, respectively. The width difference between NF-1 and NF-2 disclosed that collagen hybridizing peptides in NF-1 may adopt a single strand conformation where the collagen hybridizing peptides are fully extended with a calculated width of 25 nm assuming collagen hybridizing peptide with a polyproline-II helix as previously reported, this value is comparable to AFM measured value. Whereas collagen hybridizing peptides in NF-2 may self-trimerize and thus reduce its overall width. The AFM height profiles of both NF-1 and NF-2 were estimated to 1.2 nm, indicating the formation of a double layer anti- -sheet by (FKFE)2 (SEQ ID NO: 1). These results are consistent with previous reported results.
[00107] Although NF-1 and NF-2 are well dispersed and repel each other on their own, when mixed together, they showed strong tendency to further self-assemble into nanosheet (NS) (Figure 1). When NF-1 and NF-2 were mixed at a ratio of 1 : 1 , respectively, a large NS was observed. TEM examination of NS exhibited a well-defined assembly pattern, where NF-1 and NF-2 alternatively assembled (Figure 2E). AFM measurement also showed consistent result with TEM (Figure 2F). The AFM height profile of NS displayed an average value of approximately 1.2 nm (Figure 2F, bottom panel), which is close to the AFM height values of both NF-1 and NF-2. Taken together, these results demonstrate that NS assembly via triple helical folding of collagen hybridizing peptides are in a parallel orientation when NF-1 and NF-2 come together, and it has single-layer structure.
[00108] Since collagen hybridizing peptide has been reported to bind to denatured collagens by forming a triple-helical hybrid with the denatured collagen strands, we expected collagen hybridizing peptide-(FKFE)2 (SEQ ID NO: l) fibers can also target denatured collagen with high specificity. To investigate this, fluorescent-labeled NF-1 and NF-2 were prepared. As a control, fluorescent-labeled (FKFE)2 (SEQ ID NO: l) combined with a scrambled sequence, (FKFE)2-G9P909 (SEQ ID NO:4) fiber (NF-3), and (FKFE)2 (SEQ ID NO: 1) fiber alone (designated as NF-4) were also prepared. A gelatin-coated ELISA type plate was utilized to study the binding. The binding study performed at 37°C demonstrated that only NF-1 exhibited strong binding to gelatin, while NF-2 only showed very poor binding to gelatin (Figure 3). The controlled nanofibers did not show any affinity to gelatin, even though these nanofibers were made of highly charged amino acid residues. Since it is believed that the (FKFE)2 (SEQ ID NO: 1) peptides self-assemble into double layer anti- β-sheet wherein the hydrophobic groups are buried inside and the charged groups are neutralized, in addition to the inertness of collagen hybridizing peptides exposed outside. On the other hand, the exceptionally strong binding of NF-1 over NF-2 also confirmed that collagen hybridizing peptides in NF-1 are unfolded and fully extended outside, which makes them readily bind to denatured collagen. Whereas, collagen hybridizing peptides in NF-2 are likely presented in trimeric conformation, and thus it significantly reduce its binding affinity to denatured collagen. Further investigation on these collagen hybridizing peptide-(FKFE)2 (SEQ ID NO: 1) nanofibers, we found NF- 1 behaved similarly to gelatin. Since NF-1 coated sample showed high fluorescent intensity when CF-(GPO)9 (SEQ ID NO:5) bound to them (Figure 4). Collectively, these results indicate that NF-1 can specifically bind to denatured collagen strands and behaved similarly to gelatin, which can be exploited for various collagen hybridizing peptide-based applications.
1. Experimental section
[00109] Peptide synthesis: Peptides were synthesized on the TentaGel R RAM resin using standard F-moc chemistry using Focus XC peptide synthesizer (AAPPTec, Louisville, KY) with coupling cycles based on HBTU/DIEA-mediated (Adv Chemtech, Louisville, KY) activation. Five-fold molar excess of the amino acids and coupling reagents were used in a typical coupling reaction. The peptides were cleaved from the resins by treatment with water/l,2-ethanedithiol/thioanisole/trifluoroacetic acid
(2.5/2.5/1/94) for at least 2 hr. Peptides were purified by reversed phase-high performance liquid chromatography (RP-HPLC) (Agilent, Santa Clara, CA) on a C18 column with a gradient of water-acetonitrile containing 0.1% trifluoroacetic acid (TFA). The mass and purity of the collagen hybridizing peptides were analyzed by matrix-assisted laser desorption/ionization time-of-fiight mass spectrometry (MALDI-TOF MS)
(UltrafleXtreme, Bruker Daltonics, Billerica, MA) and HPLC (Agilent, Santa Clara, CA). The concentration of peptide solution was calculated from the weight of the dry peptide powder. 5(6)-carboxyfluorescein (CF) conjugated collagen hybridizing peptides was synthesized as reported before.
[00110] Nanofiber and nanosheet assembly: Lyophilized peptide powder samples were dissolved in water to make 2 mM solutions, followed by heating these solutions up to 80°C for 5 min. These solutions were then cooled down to 4°C for at least 2 hours, to assemble into fibers. In order to make a nanosheet, NF-1 and NF-2 were mixed together to yield a final concentration of 1 mM of each in water. This mixture was then heated up to 60°C for 5 min, followed by cooling down to 4°C for at least 2 hours.
[00111] Transmission electron microscopy (TEM): TEM was performed on FEI Tecnai T12 microscope (FEI, Hillsboro, OR) operated at 120 kV. TEM samples were prepared by applying a drop of CHP-(FKFE)2 (SEQ ID NO: l) nanofiber or nanosheet solutions 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.
[00112] Atomic force microscopy (AFM): AFM was obtained on Bruker Dimension Icon (Bruker, Billerica, MA). Five microliters sample was pipetted on a freshly cleaved mica substrate, followed by air-drying. Sample was scanned in air using tapping mode.
[00113] Circular Dichroism (CD): CD spectra were recorded on a JASCO J-1500 CD (JASCO, Tokyo) in 0.10 mm quartz cells. All CHP samples were prepared in water and incubated at 4°C for at least 24 hr prior to CD measurement. Spectra were recorded from 190 to 300 nm at a scanning rate of 100 nm/min at 0.5 nm increment. 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. Melting temperatures were determined from the maximum of the first derivative of the melting curves. [00114] Gelatin binding assay: Gelatin binding assay was performed by using an ELISA type plate. An ELISA type plate was prepared by coating with 0.5% gelatin solution at 4°C for 2 hours, with gentle shaking, then washed three times with PBS buffer, followed by blocking with 5% BSA at RT for 1 hour, washed three with PBS buffer prior to assay. Ten μΜ of each fluorescent-labelled nanofiber was incubated with gelatin coated plate at 37°C for 2 hours, then washed three times with 37°C PBS buffer. SpectraMax M-2 microplate reader was used to measure the fluorescence (ex: 489 nm, em: 533 nm). Each binding experiment was done in triplicate.
[00115] FTIR Spectroscopy. FTIR spectroscopy measurement was acquired using a Nicolet iZIO Fourier transform infrared spectrometer (Thermo Scientific). Solution samples were freeze-dried into powder. The powder and potassium bromide were compressed into a thin pellet. Data were fitted using Gaussian function.
[00116] X-ray diffraction (XRD): Nanofibers/nanosheet solution samples were freeze- dried into powder, and loaded into capillary tubes. X-ray diffraction images were collected on a Rigaku Micromax-007 X-ray generator equipped with an R-Axis IV++ area detector.
2. Applications for collagen hybridizing peptide-(FKFE)2 (SEQ ID NO:l) nanofibers and nanosheet:
[00117] Collagen hybridizing peptide-(FKFE)2 (SEQ ID NO: 1) nanofibers and nanosheets can be used for staining tissue, ELISAs, tissue engineering, targeting and delivering systems, promoting T cell activity, and material sciences.
[00118] Staining tissue: collagen hybridizing peptide-nanofibers can be used to amplify the signals, since they can carry large numbers of molecules in one fiber.
[00119] Developing ELISA type detection method: The same type collagen hybridizing peptide-nanofibers are well dispersed and repel each other on their own, which can be applied to develop an ELISA type assay to detect diseased collagen fragments in body fluid. In this assay, an ELISA plate can firstly coat with collagen hybridizing peptide- nanofiber, followed by applying the sample on it, and finally detect with the same type collagen hybridizing peptide-nanofiber conjugated with signal molecules (ex.
Fluorescence, biotin, ect).
[00120] Developing scaffold for tissue engineering: collagen hybridizing peptide- nanofiber conjugated with therapeutic molecules can directly electron-spray with gelatin to make a gelatin-modified scaffold for would healing and tissue regeneration
applications. [00121] Targeting and delivering systems: Collagen hybridizing peptide-nanofiber can bind to denatured collagen, and most importantly, are very soluble and well-dispersed in buffer. Collagen hybridizing peptide-conjugated with therapeutic molecules can also target and deliver large numbers of therapeutic molecules to collagens undergoing degradation in vivo.
[00122] Promoting T cell activity: When combined with collagen hybridizing peptides, the (FKFE)2 (SEQ ID NO: l) nanofiber can become very inert, well-dispersed and highly soluble in buffer. Utilizing their dimensionally continuous structures, these nanofibers and nanosheets can be further engineered to tether large numbers of bio-macromolecules (such as, antibodies and proteins) to promote T cell activity. Nanosheets can also be used for clustering the T cell while stimulating them in T cell therapeutic applications.
[00123] Impact of controlling the nanosheet formation in material sciences: Although significant research effort has been focused on creating complex materials based on self- assembly, the focus is mainly on one-dimensional materials such as nanofibers, nanotube, nanowire, etc. There are limited works on two-dimensional nanosheets. It still remains many challenges to fabricate such materials. So far, DNA nanotechnology, based on defined molecular recognition of Waston-Crick base pairing rules, has enabled researchers to create various dimensionally-controlled materials ranging from 1-D, 2-D to 3-D assemblies. Whereas controlling protein-protein interaction is much more challenging due to its high degree of complexity, and unpredictability. However, the unique recognition and self-assembly properties of peptides offers many advantages that allow us to control the formation of many defined structures (nanofibers, nanotubes, nanowires, etc), in addition to its modifiable ability, chemical stability, and inertness, as well as ease of large scale production.
[00124] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS We claim:
1. A peptide conjugate comprising
a. a (FKFE)2 beta-sheet forming peptide;
b. a spacer moiety; and
c. a collagen hybridizing peptide.
2. The peptide conjugate of claim 1 , wherein the collagen hybridizing peptide comprises the sequence of at least (GXY)n, wherein G is glycine, wherein X and Y are any amino acid, and wherein n is at least 3.
3. The peptide conjugate of claim 2, wherein X is proline, glutamic acid, or aspartic acid.
4. The peptide conjugate of any one of claims 2-3, wherein Y is a modified proline, lysine, or arginine.
5. The peptide conjugate of claim 4, wherein the modified proline is hydroxyproline or fluoroproline.
6. The peptide conjugate of any one of claims 2-5, wherein a glycine is modified as an aza-glycine.
7. The peptide conjugate of any one of claims 2-5, wherein a lysine is modified as an pyrrolysine.
8. The peptide conjugate of any one of claims 1-7, further comprising an active agent.
9. The peptide conjugate of claim 8, wherein the active agent is a detectable moiety or a therapeutic agent.
10. The peptide conjugate of claim 9, wherein the detectable moiety is a fluorescent dye, radioactive isotope, magnetic bead, metallic bead, colloidal particle, near- infrared dye, or an electron-dense reagent.
11. The peptide conjugate of any one of claims 8-10, wherein the active agent is attached to the N-terminal or C-terminal portion of the peptide conjugate.
12. The peptide conjugate of any one of claims 1 -11 , wherein the spacer moiety is between the (FKFE)2 beta-sheet forming peptide and the collagen hybridizing peptide.
13. The peptide conjugate of any one of claims 1 -12, wherein the spacer moiety comprises at least three glycines or aminohexanoic acid.
14. The peptide conjugate of claim 9, wherein the therapeutic agent is a therapeutic known to treat a disease or injury involving collagen damage.
15. The peptide conjugate of claim 14, wherein the therapeutic agent is an antibody, a peptide, a nucleic acid, or a chemical compound.
16. The peptide conjugate of claim 14, wherein the disease or injury is cancer, osteoporosis, arthritis, fibrosis, or a chronic wound.
17. The peptide conjugate of any one of claims 1 -16, wherein the collagen hybridizing peptide is on either the N-terminal end or C-terminal end of the (FKFE)2 beta- sheet forming peptide.
18. The peptide conjugate of any one of claims 1 -17, wherein the collagen hybridizing peptide is a dimeric collagen hybridizing peptide.
19. A composition comprising one or more of the peptide conjugates of any one of claims 1 -18.
20. The composition of claim 19, further comprising a pharmaceutically acceptable carrier.
21. A nanofiber comprising two or more of the peptide conjugates of any one of claims 1 -18.
22. A nanosheet comprising two or more of the nanofibers of claim 21.
23. The nanosheet of claim 22, wherein the two or more nanofibers are opposite orientations.
24. A method of detecting denatured collagen in a sample comprising
a. contacting a composition comprising the peptide conjugate of any one of claims 1 -18, and
b. detecting the presence or absence of binding of the peptide conjugate to denatured collagen, wherein the presence of binding indicates the presence of denatured collagen.
25. A method of treating a disease or injury involving collagen damage comprising administering to a subject having a disease or injury involving collagen damage a peptide conjugate of any one of claims 1 -18.
26. The method of claim 25, wherein the disease or injury is cancer, osteoporosis, arthritis, fibrosis, or a chronic wound.
27. A method of screening for a therapeutic agent that treats a disease or injury involving collagen damage comprising
a. detecting the presence or absence of binding of a peptide conjugate to denatured collagen, the presence of binding indicating the presence of denatured collagen in the sample; b. administering to a sample having denatured collagen a composition comprising the peptide conjugate of claim 9; and
c. detecting the presence or absence of binding of the peptide conjugate to denatured collagen, the presence of binding indicating the presence of denatured collagen in the sample,
wherein a decrease in the amount of damaged or denatured collagen in the sample detected after administration of the peptide conjugate compared to the amount detected prior to administration of the peptide conjugate indicates a therapeutic agent capable of a disease or injury involving collagen damage.
28. A kit comprising
a. a (FKFE)2 beta-sheet forming peptide;
b. a spacer moiety; and
c. a collagen hybridizing peptide.
PCT/US2017/019724 2016-12-06 2017-02-27 Collagen targeting nanofibers and nanosheets WO2018106273A1 (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111407727A (en) * 2020-03-26 2020-07-14 浙江大学 Application of mammal target of rapamycin (mTOR) blocker in preparation of medicines for treating calcified tendinosis
CN111704663A (en) * 2020-06-29 2020-09-25 江南大学 Preparation method of collagen hydrogel
WO2023034903A1 (en) * 2021-09-01 2023-03-09 3Helix, Inc. Systems and methods for detecting fibrosis
CN115925988A (en) * 2022-09-14 2023-04-07 中山大学附属第五医院 Modified collagen targeted antibacterial peptide and preparation method and application thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080287342A1 (en) * 2004-11-23 2008-11-20 The Johns Hopkins University Compositions Comprising Modified Collagen and Uses Therefor
US20130115196A1 (en) * 2010-03-16 2013-05-09 Escape Therapeutics, Inc. Hybrid hydrogel scaffold compositions and methods of use
WO2016123570A1 (en) * 2015-01-30 2016-08-04 University Of Utah Research Foundation Dimeric collagen hybridizing peptides and methods of using

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080287342A1 (en) * 2004-11-23 2008-11-20 The Johns Hopkins University Compositions Comprising Modified Collagen and Uses Therefor
US20130115196A1 (en) * 2010-03-16 2013-05-09 Escape Therapeutics, Inc. Hybrid hydrogel scaffold compositions and methods of use
WO2016123570A1 (en) * 2015-01-30 2016-08-04 University Of Utah Research Foundation Dimeric collagen hybridizing peptides and methods of using

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111407727A (en) * 2020-03-26 2020-07-14 浙江大学 Application of mammal target of rapamycin (mTOR) blocker in preparation of medicines for treating calcified tendinosis
CN111407727B (en) * 2020-03-26 2021-03-23 浙江大学 Application of mammal target of rapamycin (mTOR) blocker in preparation of medicines for treating calcified tendinosis
CN111704663A (en) * 2020-06-29 2020-09-25 江南大学 Preparation method of collagen hydrogel
WO2023034903A1 (en) * 2021-09-01 2023-03-09 3Helix, Inc. Systems and methods for detecting fibrosis
CN115925988A (en) * 2022-09-14 2023-04-07 中山大学附属第五医院 Modified collagen targeted antibacterial peptide and preparation method and application thereof
CN115925988B (en) * 2022-09-14 2023-08-22 中山大学附属第五医院 Denatured collagen targeted antibacterial peptide and preparation method and application thereof

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