A REVIEW of the HIGHER-ORDER STRUCTURES and APPLICATIONS of COLLAGEN MIMETIC PEPTIDES a Thesis Presented to the Graduate Facul

A REVIEW OF THE HIGHER-ORDER STRUCTURES AND APPLICATIONS OF

COLLAGEN MIMETIC PEPTIDES

A Thesis

Presented to

The Graduate Faculty of The University of Akron

In Partial Fulfillment

of the Requirements for the Degree

Master of Polymer Science

Max Goldmeier

May 2016 A REVIEW OF THE HIGHER-ORDER STRUCTURES AND APPLICATIONS OF

COLLAGEN MIMETIC PEPTIDES

Max Goldmeier

Thesis

Approved: Accepted:

______Faculty Advisor Dean of the College Dr. Nita Sahai Dr. Eric J. Amis

______Faculty Co-Advisor Dean of the Graduate School Dr. William J. Landis Dr. Chand Midha

______Department Chair Date Dr. Coleen Pugh

ABSTRACT

Collagen is a protein that is a major component of the human body and makes up thirty percent of the total protein in the human body. Collagen mimetic peptides (CMPs) have been used to study the formation of the triple helix of collagen and its higher- ordered structures. This article discusses how CMPs have been used to learn about the physical properties of the triple helix and how the amino acid sequence affects the physical properties, formation, and stability of the triple helix. CMPs have also been designed to form higher-ordered structures for various applications. This work reviews the design principles and strategies employed with various interactions to form higher- ordered structures from CMPs. These strategies include the use of electrostatic interactions, the incorporation of cysteine knots, and metal-induced self-assembly, among others. Many of these higher-ordered structures have applications such as biomolecule attraction, cellular adhesion, nanowire fabrication, and hydrogel formation.

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DEDICATION

This work is dedicated to my family for their support and encouragement.

ACKNOWLEDGEMENTS

I would like to thank Dr. Nita Sahai and Dr. William Landis from the Department of Polymer Science at The University of Akron in Akron, OH for their guidance and support which made this work possible.

TABLE OF CONTENTS

Page

LIST OF TABLES…………………………………………………………………...…viii

LIST OF FIGURES………………………..……………………...... ix

CHAPTER

I. INTRODUCTION……………………………………………………..………………..1

1.1 Introduction………………………………………………...………………….1

1.2 Features of Collagen and Biosynthetic Process of Collagen Formation………5

II. HELICAL TWIST OF THE TRIPLE HELIX OF COLLAGEN…………………….10

III. TRIPLE HELIX STABILITY AND AMINO ACID SEQUENCE… ………….…..13

3.1 Introduction to Collagen Triple Helix Stability……………………………...13

3.2 Tertiary Amides……………………………………………………………...14

3.3 Stereoelectronic Effects……………………………………………………...15

3.4 Hydrogen Bonding…………………………………………………………...17

3.5 Electrostatic Interactions……………………………………………………..18

3.6 The Role of Glycine in Triple Helix Stability……………………………….19

3.7 Conclusion of Triple Helix Stabilizing Factors……………………………...21

IV. HIGHER-ORDER CMP STRUCTURES………………...... ………..22

4.1 Intro to Higher-Order Structure and Applications of CMPs…………………22

4.2 Pro-Hyp-Gly CMPs………………………………………………………….23

TABLE OF CONTENTS (CONT.)

Page

4.3 Pro-Pro-Gly CMPs…………………………………………………………...24

4.4 CMP Amphiphiles…………………………………………………………...29

4.5 2+1 Strand Click Synthesis…………………………………………………..34

4.6 Cysteine Bridges……………………………………………………………..36

4.7 Aromatic Interactions………………………………………………………...38

4.8 Metal-Induced Self-Assembly……………………………………………….41

4.9 Electrostatic Interactions……………………………………………………..47

4.10 Nanodiamond-CMP Conjugates……………………………………………52

4.11 Polymerized-CMPs and CMP-Dendrimers…………………………………52

V. CONCLUSION ………………………………………………………………………59

VI. BIBLIOGRAPHY……………………………...…………………………………….61

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LIST OF TABLES

Table Page 1. Chart displaying the types of collagen, their classification, distribution, and pathology.2, 3………………………………………………………………….……2

2. The amino acid composition of type I collagen α1and α2 chains that form the type I collagen triple helix. The Pro-Hyp-Gly sequence (the corresponding amino acids are in bold) is the highest occurring sequence in the chains.1 Amino acids are organized by highest content in the α1(I) chain to lowest………………………...6

3. The stability effects of tertiary amides in comparison to POG and PPG sequences. Tertiary amides other than Nleu tend to have a destabilizing effect on the Tm of CMP triple-helices……………………………………………………………….14

4. Stereoelectronic effects contribute to the stability of the triple helix as shown by the use of fluorinated derivatives of proline. Tm values from Ref. 2……………16

5. Acp and Fmp were used to show that even though a residue may not have the appropriate ring puckering for the position it is in, the hydrogen bonding network can provide stability to the triple helix. Tm values from Ref. 45………………...18

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LIST OF FIGURES

Figure Page

1. Schematic of the biosynthesis of collagen. Genetic coding for collagen is transcribed to form a protocollagen strand. The protocollagen strand undergoes enzymatic modification to form a procollagen triple helix. Proteinases trim the N- and C-termini thus producing a tropocollagen triple helix which self-assembles into fibrils that cross-link to form fibers.2, 6 Reprinted with permission from Shoulder, MD, Raines, RT, Annual Review of Biochemistry, 2009, 78, 929-958 © 2009, Annual Reviews………………………………………………………….7

2. TEM and AFM images of collagen displaying D-periodicity. A) TEM of collagen fibrils from human Achilles tendon stained with gold-palladium. Scale bar = 500 nm. B) TEM of type I collagen from calk-skin stained with phosphotungstic acid. Scale bar = 100 nm. C) AFM image of corneal collagen fibrils. Scale bar = 100 nm. D) AFM image of scleral collagen fibrils. Scale bar = 100nm. A, B) Adapted with permission from Electron Microscopy Reviews, 3/1, Chapman, JA, et al., The collagen fibril – a model system for studing the staining and fixtion of a protein, 143-182 © 1990 with permission from Elsevier. C, D) Adapted with permission from Japanese Journal of Ophthalmology, 46/5, Yamamto, S, Hitomi, J, et al., Observation of human corneal and scleral collagen fibrils by atomic force microscopy, 496-501 © 2002 with permission from Elsevier…………………….8

3. A scheme of the helical twist and unit height parameters. In a 72 helical twist, there are 21 amino acids that constitute two turns of the helix and a unit height of 20 Å. Reprinted with permission from Biopolymers, 84/4, Okuyama, K, Wu, G, et al., Helical twists of collagen model peptides, 421-432 © 2006 John Wiley and Sons………………………………………………………………………………11

4. Cϒ-endo and Cϒ-exo ring puckering of the pyrrolidine ring residues. Proline or proline derivatives with Cϒ-endo ring puckering in the X position and Cϒ-exo ring puckering in the Y position provide stability to the collagen triple helix through stereoelectronic effects. These stereoelectronic effects orient the main chain dihedral angles of the collagen chain to form a triple helix…………….………..15

LIST OF FIGURES (CONT.)

Figure Page

5. A schematic diagram of a one-residue stagger and interchain hydrogen bonding between three strands (dashed line) that enables triple helix formation in procollagen and (POG)n CMPs…………………………………………………..17

6. CMP design with a Gly→Ala substitution.49 This CMP mimics the defective collagen found in patients suffering from OI…………………………………….20

7. TEM of (POG)10 and polyPOG. A) (POG)10 with branched extensive branching of 64 65 the fibers. B) (POG)10 aggregates of nanofibers. C) poly-POG fibers 65 displaying higher dispersion of fibers than (POG)10. A) Reprinted with permission from The Journal of Biological Chemistry, 281/44, Kar, K, Amin, P, et al., Self-association of collagen triple helic peptides into higher order structures, 33283-33290 © 2006 American Society for Biochemistry and Molecular Biology B, C) Adapted with permission from Biopolymers, 79/3, Kishimoto, T, Morihara, Y, et al., Synthesis of poly (Pro–Hyp–Gly)n by direct polycondensation of (Pro–Hyp–Gly)n, where n= 1, 5, and 10, and stability of the triple‐helical structure, 163-172 © 2005 John Wiley and Sons……………….…25

8. TEM of nanofibers from a CMP with the sequence (GPP)3GPR-GEKGER- 72 GPR(GPP)3-GPCCG at different scales. A, B) Scale bar = 0.5 μm and C, D) Scale bar = 1μm. Reprinted with permission from Biomacromolecules, 32/7, Krishna, O. D., and Kiick, K. L., Integrin-mediated adhesion and proliferation of human MSCs elicited by a hydroxyproline-lacking, collagen-like peptide, 6412- 6424 © 2009 American Chemical Society...... …26

9. A) Schematic of PPG CMP F877. The underlined G at position 901 was substituted with Ser in the G901S CMP. B) TEM of F877 nanofibers. Scale bar = 40 nm. C) TEM of G901S fibers. Scale bar = 40nm. Adapted with permission from Angewandte Chemie International Edition, 46/18, Bai, H, Xu, K, et al., Fabrication of Au nanowires of uniform length and diameter using a monodisperse and rigid biomolecular template: collagen‐like triple helix, 3383- 3386 © 2007 John Wiley and Sons…………………………...………………….27 x

LIST OF FIGURES (CONT.)

Figure Page

10. A) TEM of the cubes assembled by streptavidin-labelled gold nanoparticles and a biotinylated F877 CMP. Scale bar = 1μm. B) TEM of the hexagonal structures that assembled when the diameter of the gold nanoparticles was raised to 30 nm. Scale bar = 1μm. Adapted with permission from Angewandte Chemie International Edition, 49/45, Kaur, P, Maeda, Y, et al., Three‐dimensional directed self‐assembly of peptide nanowires into micrometer‐sized crystalline cubes with nanoparticle joints, 8375-8378 © 2010 John Wiley and Sons……....28

11. A) Schematic of the B877B CMP. B) Schematic of the process of forming microcapsules on the octane/water interface. Streptavidin was used to lock the structure of the microcapsules. C) Illustration of the assembled structure of the microcapsules. D) Fluorescence micrograph of the microcapsules. Streptavidin- stabilized microcapsules have a higher contrast. E) TEM of B877B microcapsules when streptavidin was bound to quantum dots. The inset image is a magnified image of the QD-streptavidin-B877B microcapsules. Adapted with permission from Small, 8/9, Maeda, Y, Wei, Z, Matsui, H, Biomimetic assembly of proteins into microcapsules on oil‐in‐water droplets with structural reinforcement via biomolecular‐recognition‐based cross‐linking of surface peptides, 1341-1344 © 2012 John Wiley and Sons……………………………………………………….29

12. Structure of the IV-H1 CMPA83 where the 15-residue guest sequence mimics collagen sequence α1(IV)1263-1277. Melanoma cells were capable of adhesion and spreading on surfaces coated with the IV-H1 CMP.89…...………….………31

13. A) Schematic of α1(IV)1263-1277 CMPA sequence and the structure of its triple helix. B) The assembled liposome loaded with a fluorophore. C) The CMPA binds with the liposome by inserting the hydrophobic tail onto the bilayer of the liposome. D) The CMPA epitope segment binds with the CD44 receptors of a melanoma cell and releases the cargo from the liposome. Reprinted with permission from Journal of the American Chemical Society, 129/16, Rezler, EM, Khan, DR, et al., Targeted drug delivery utilizing protein-like molecular architecture, 4961-4972 © 2007 American Chemical Society…………………..32

LIST OF FIGURES (CONT.)

Figure Page

14. A, B) TEM of CMPA container a GFOGER integrin-binding guest sequence. These fibers have a ~16nm diameter. A) Scale bar = 200nm. B) Scale bar = 50 nm. C) The CMPA was able to form a nanofibrous hydrogel. D) The vial containing the hydrogel was inverted to show that the hydrogel is self- supporting.96 Adapted with permission from ACS Nano, 5/10, Luo, J, and Tong, YW, Self-assembly of collagen-mimetic peptide amphiphiles into biofunctional nanofiber, 7739-7747 © 2011 American Chemical Society……………………..33

15. A) An overall schematic showing the PA self-assembled into a nanofiber and then underwent the self-templating process and was pulled to form a self-tamplated pattern on a surface. B) TEM image of a nanofiber. Scale bar = 50 nm. C) AFM image of nanofibers. Scale bar = 1 μm. D) Self-tamplating yielded a twist plywood pattern with vertical periodic spacing. Top - scale bar = 50 μm. Bottom – scale bar = 20 μm. E) Crimp pattern achieved from the self-templating process with a periodic horizontal pattern. Top – scale bar = 40 μm. Bottom – scale bar = 20 μm. F) Scheme showing MC3T3-E1 cells and HA were seeded ontp the pattern scaffold. G) Bright field and fluorescent microscopy images of MC3T3- E1 preosteoblast cells that were cultured for 5 days on a self-templated surface aligned with the grooves (scale bar = 100 μm, actin in green, nuclei in blue). Adapted with permission from Nano Letter, 15/10, Jin, HE, Jang, J, et al., Biomimetic self-templated hierarchical structures of collagen-like peptide amphiphiles, 7138-7145 © 2015 American Chemical Society………………….34

16. Schematic diagram of Byrne et al.100 for 2+1 strand click synthetic chemistry. Reprinted with permission from Chemical Communications, 47/9, Byrne, C, McEwan, PA, et al., End-stapled homo and hetero collagen triple helices: a click chemistry approach, 2589-2591 © 2010 with permission of The Royal Society of Chemistry………………………………………………………………………...36

17. Scheme of the triple helix of a heterotrimeric POG CMP.103 Cysteine knots (green) gave researchers the ability to add stability to the triple helix. The cysteine knots also enabled the ability to form a heterotrimeric CMP……………………37

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LIST OF FIGURES (CONT.)

Figure Page

18. Schematic for the assembly of a hydrogel from the self-assembly of staggered triple helices. Reprinted with permission from Biopolymers Peptide Science, 90/6, Yamazaki, CM, Asada, S, et al., Artificial collagen gels via self‐assembly of de novo designed peptides, 816-823 © 2008 John Wiley and Sons………………...38

19. The design schematic of the POG based CMP with either GFOGER or GPOGER integrin-binding sequences.106 The triple helices self-assemble into nanofibers and were capable of cell adhesion. Reprinted with permission from Biopolymers, 31/7, Yamazaki, CM, Kadoya, Y, et al., A collagen-mimetic triple-helical supramolecule that evokes integrin-dependent cell responses, 1925-1934 © 2010 Elsevier…………………………………………………………………………..39

20. A) Chemical structure of the CMP from Ref. 107. B) Image showing the interactions of aromatic interactions of peptides in the triple helix. C) The CMP is capable of aggregating platelets similarly to collagen. D) TEM of CMP nanofibers. E) TEM of a cross-section of a murine blood vessel. Adapted with permission from the Journal of the American Chemical Society, 129/8, Cejas, MA, Kinney, WA, et al., Collagen-related peptides: self-assembly of short, single strands into a functional biomaterial of micrometer scale, 2202-2203 © 2007 American Chemical Society……………………………………………………..40

21. Chemical structure of the CMP containing phenylalanine and pentaflourophenyl termini groups and schematic to yield gold-labelled nanowires. B) TEM of CMP fibers before electroless silver plating. C) TEM of CMP fibers after electroless silver plating. D) TEM of CMP coated with silver nanoparticles after one deposition and one hour. Adapted with permission from the Journal of Materials Chemistry, 18/32, Gottlieb, D, Morin, SA, et al., Self-assembled collagen-like peptide fibers as templates for metallic nanowires, 3865-3870 © 2008 with permission of The Royal Society of Chemistry………………………………….41

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LIST OF FIGURES (CONT.)

Figure Page

22. A) Chemical structure of the KByp-containing CMP from Ref 113. B) The peptide strands self-assemble into a triple helix. C) Upon the addition of Fe(II), the triple-helices grow in the radial direction to form fibers. D) TEM image of the fibers. Adapted with permission from the Journal of the American Chemical Society, 130/38, Przybyla, DE, and Chmielewski, J, Metal-triggered radial self- assembly of collagen peptide fibers, 12610-12611 © 2008 American Chemical Society……………………………………………………………………………43

23. A) Chemical structures of HisCol and IdaCol CMPs from Ref. 61. B) The peptide strands self-assemble into triple helices and adding metals binds the two CMPs. C-E) SEM images of the assemblies formed from C) Ni(II), scale bar = 1 μm D) Zn(II), scale bar = 500 nm E) Cu(II), scale bar = 500 nm. F) TEM images of the higher-ordered structures, scale = 700nm and insets at a higher magnification. G) TEM of the periodic banding suggests the length of the triple helices is 9 nm. Adapted with permission from the Journal of the American Chemical Society, 133/37, Pires, MM, Przybyla, DE, et al., Metal-mediated tandem coassembly of collagen peptides into banded microstructures, 14469-14471 © 2011 American Chemical Society………………………………………………………………...44

24. A) Structure of the NTA CMP.115 B-E) SEM images of B) microflorettes, scale bar = 10 μm and C-E) saddle structures, scale bar = 3 μm. Adapted with permission from Langmuir, 28/4, Pires, MM, Lee, J, et al., Controlling the morphology of metal-promoted higher-ordered assemblies of collagen peptides with varied core lengths, 1993-1997 © 2012 American Chemical Society……..45

25. A) Chemical structure of the catechol-containing CMP from Ref. 120. The CMP forms a triple helix due to the ethylenediamine scaffold. B) Upon the addition of Fe3+ the stability of the triple helix was raised by 22 °C. Adapted with permission from the Journal of the American Chemical Society, 126/46, Cai, W, Kwok, SW, et al., Metal-assisted assembly and stabilization of collagen-like triple helices, 15030-15031 © 2004 American Chemical Society……………………………...47

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LIST OF FIGURES (CONT.)

Figure Page

26. A) Structure of the (PRG)4-(POG)4-(EOG)4 CMP. B) TEM image of fibers formed from the CMP with pointed tips. C) TEM displaying the 18 nm D- periodicity of the CMP fibers. D) TEM of the tactoidal end (circled) of a collagen fibril. A-C) Adapted with permission from the Journal of the American Chemical Society, 129/47, Rele, S, Song, Y, et al., D-periodic collagen-mimetic microfibers, 14780-14787 © 2007 American Chemical Society D) Adapted with permission from the Journal of Biological Chemistry, 254/22, Gelman, RA, Poppke, DC, and Piez, KA, Collagen fibril formation in vitro. The role of the nonhelical terminal regions, 11741-11745 © 1979 American Society for Biochemistry and Molecular Biology……………………………………………48

27. A) TEM of the NSI CMP showing the uneven distribution of the sizes of the nanosheets. Scale bar = 1 μm B) TEM of an individual nanosheet from NSI. Scale bar = 1 μm. C) TEM of an NSII nanosheet Scale bar = 500 nm. D) AFM image of an NSI nanosheet that has multiple layers. Scale bar = 200nm. E) AFM of an NSII single layer nanosheet. Scale bar = 200 nm. F) AFM height histogram of single-layered sheets of NSI (black) and NSII (white). Reprinted with permission from the Journal of the American Chemical Society, 136/11, Jiang, T, Xu, C, et al., Structurally defined nanoscale sheets from self-assembly of collagen-mimetic peptides, 4300-4308 © 2014 American Chemical Society………………………50

28. A) TEM of a single detonation ND particle approximately 5 nm in diameter with an inert sp3 diamond core. B) 5 nm diameter atomistic model of a ND displaying the many functional groups present on the surface that make selectivity in synthesis a challenge. Grey: sp3 diamond carbon. Green: sp3 amorphous carbon. Black: sp2 carbon. Red: oxygen. Blue: nitrogen. White: hydrogen. Adapted with permission from Biopolymers Peptide Science, 104/3, Knapinska, AM, Tokmina- Roszyk, D, et al., Solid‐phase synthesis, characterization, and cellular activities of collagen‐model nanodiamond‐peptide conjugates, 186-195 © 2015 John Wiley and Sons…………………………………….…………………………………....53

LIST OF FIGURES (CONT.)

Figure Page

29. CMP-polymer designed by Hartgerink and coworkers using native chemical ligation.132 A) Schematic of the mechanism of native chemical ligation showing the intermediate thioester formed from the N-terminal cysteine. B) The N- terminal cysteine and C-terminal thioester polymerize through native chemical ligation. Two reactive groups are shown in the squares. C) To control the polymerization, hydrolysis of the thioester group prevents the polymer chain from growing uncontrollably. Reprinted with permission from Macrmolecules, 38/18, Paramanov, SE, Gauba, V, and Hartgerink, JD, Synthesis of collagen-like peptide polymers by native chemical ligation, 7555-7561 © 2005 American Chemical Society……………………………………………………………………………55

30. A, B) In a PEODA hydrogel, chondrocytes secrete collagen, however in the CMP/PEODA hydrogel, interactions between the CMP and collagen enabled the hydrogel to retain cell-secreted collagen and promoted a fast accumulation of extracellular matrix products.135 C) Fluorescence micrograph of chondrocytes embedded in the CMP/PEODA hydrogel. Adapted with permission from Biomaterials, 27/30, Lee, HJ, Lee, JS, et al., Collagen mimetic peptide-conjugated photopolymerizable PEG hydrogel, 5268-5276, © 2006 Elsevier………………56

31. A) Schematic showing the addition of CMP to an 8-arm star poly(ethylene glycol) to form a hydrogel for the encapsulation of human mesenchymal stem cells. The insert is an SEM image that shows the porous structure of the hydrogel. B, C) SEM of the hydrogel at two different magnifications. D) Fluorescence image of the human mesenchymal stem cells encapsulated in the hydrogel. E) Cryo-SEM image of the hMSCs in the hydrogel. Reprinted with permission from Macromolecular Bioscience, 11/10, Rubert Perez, CM, Panitch, A, and Chmielewski, J, A collagen peptide‐based physical hydrogel for cell encapsulation, 1426-1431 © 2011 John Wiley and Sons………………………..57

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LIST OF FIGURES (CONT.)

Figure Page

32. Schematic showing the structure of the TRISA-CMP and the three triple helices bound around a trimesic acid core.137 Reprinted with permission from the Journal of the American Chemical Society, 124/51, Kinberger, GA, Cai, W, and Goodman, M, Collagen mimetic dendrimers, 15162-15163 © 2002 American Chemical Society……………………….…..……………………………………58

33. A) Schematic showing the preparation of PAMAM dendrimer-bound CMPs containing the GFOGER sequence.140 B-D) Images of the actin cytoskeletal structure of Hep3B cells on B) calf-skin collagen, C) enzymatically crosslinked CMD-Q and CDM-K CMPs and on D) CDM-K. Adapted with permission from Biomaterials, 29/20, Khew, ST, Yang, QJ, and Tong, YW, Enzymatically crosslinked collagen-mimetic dendrimers that promote integrin-targeted cell adhesion, 3034-3045 © 2008 Elsevier…………………………………………...59

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CHAPTER 1

INTRODUCTION

1.1 Introduction

Collagen is a triple-helical protein widely found in animals, is a third of the total protein content in humans, and is also a major component of the extracellular matrix in humans. Collagen is forms higher-order structures such as fibers and networks that are critical to its functions. Collagen-based biomaterials, such as hydrogels and sponges, have been used for many medical treatments, including sutures, ligamentary prostheses, and wound dressings.1 The triple helix of collagen is composed of three left-handed peptide strands that are parallel to each other and feature a polyproline type II helical conformation.2, 3 The three strands coil around each other to form a right-handed triple helix.2, 3 The three strands in the triple helix have a one residue stagger.2-4 There are 28 different types of collagen and they are organized into five categories: fibril-forming, network-forming, fibril associated collagens with interrupted triple helices (FACITs), membrane-associated collagens with interrupted triple helices (MACITs), and multiple 1

triple helix domains and interruptions (MULTIPLEXINS) (Table 1).2, 3, 5 Each of the 28 types of collagen have distinct functions and some types of collagen are found in tandem with other types of collagen in a specific tissue (Table 1).2, 3 For example, type XII collagen is colocalized with type I collagen in the human skin and tendons.2, 3

Collagen is a large protein with over 1000 amino acids is not soluble in water thus making it a challenging protein to study. For this reason, smaller collagen-mimetic peptides (CMPs) have been used to elucidate the structural features of collagen. CMPs are generally composed of 30 amino acids which makes them feasible to study. The Pro-

Hyp-Gly (POG) sequence was most commonly studied because of its frequent occurrence in collagen (Table 2).1 Many other collagen-mimetic sequences have also been studied to gain insight into how specific collagenous sequences contribute to the stability and biological roles of collagen.

This paper can be divided into three sections. The first chapter discusses the features, characteristics, and biosynthesis of collagen. Although a few types of collagen are discussed in this work, the vast majority of the focus is on type I collagen and on type

III collagen. The second section of this paper, comprised of chapters 2 and 3, focuses on how CMPs have been used to learn the effect of the specific amino acid sequence on the stability of the triple helix of both collagen and CMPs. Chapter two discusses how the amino acid sequence effects the helical twist of a triple helix, meaning how many amino acid residues form a twist of the triple helix and how tight or loose that triple helix is

Collagen Type Classification Distribution / Pathology Bone, ligament, tendon, dermis / OI, osteoporosis, Ehlers- I Fibril-forming Danlos syndrome II Fibril-forming Cartilage, vitrous / osteoarthrosis, chondrodysplasias Intestine, blood vessels, skin, hollow organs, Colocalized III Fibril-forming with collagen I / Ehlers-Danlos syndrome, arterial aneurysms IV Network-forming Basement membranes / Alport syndrome Dermis, bone, placenta, cornea, Colocalized with collagen V Fibril-forming I / Ehlers-Danlos syndrome Muscle, cartilage, bone, dermis, cornea / Bethlem VI Beaded-filament-forming myopathy Bladder, dermis, dermal-epidermal junction / VII Anchoring fibrils epidermolysis bullosa acquisita Kidney, brain, heart, dermis, Descement’s membrane / VIII Network-forming Fuchs endothelia cornial dystrophy Colocalized with collagen II, cornea, cartilage, vitreous / IX FACIT osteoarthrosis, multiple epiphyseal dysplasia X Network-forming Cartilage / chondrodysplasia Colocalized with collagen II, intervertebral disk, cartilage / XI Fibril-forming chondrodysplasia, osteoarthrosis XII FACIT Colocalized with collagen I, tendon, dermis XIII MACIT, Transmembrane Eye, dermis, heart, endothelial cells XIV FACIT Colocalized with collagen I, cartilage, bone dermis XV MULTIPLEXIN Testis, capillaries, heart, kidney, eye, muscle, microvessels XVI FACIT Kidney, dermis Epithelial hemidesmosomes / generalized atrophic XVII Transmembrane epidermolysis bullosa Liver, basement membranes, important for retinal XVIII MULTIPLEXIN vasculogenesis / Knobloch syndrome XIX FACIT Basement membranes XX FACIT Cornea XXI FACIT Kidney, stomach XXII FACIT Localized at tissue junctions XXIII MACIT, Transmembrane Retina, heart XXIV Fibril-forming Cornea, bone XXV MACIT, Transmembrane Testis, heart, brain XXVI Beaded-filament-forming Ovary, testis Embryonic and adult cartilage, dermis, cornea, retina, XXVII Fibril-forming heart arteries XXVIII Beaded-filament-forming Sciatic nerve, dermis

Table 1. Chart displaying the types of collagen, their classification, distribution, and pathology.2, 3

3 wound. Chapter three discusses how the specific amino acids alter the melting temperature of CMPs, at which the triple helix begins to unravel. The topics covered in chapters 1, 2, and 3 have already been extensively reviewed and are covered in this workbecause they are the cornerstone and fundamentals of CMP research. These sections have basis in previous reviews and they are cited as such, specifically references 1 – 5.

Chapter 4 is the focus of this work and discusses the higher-order structures and applications of CMPs. This review provides an extensive and comprehensive coverage of the higher-order structures and applications of CMPs in a way that has not been done before. Great reviews have been written but either they focus on a narrower range, have a different global focus, or just aren’t very current. Also, it is important to note that the term ‘higher-order structures and applications’ is used here quite liberally. Higher-order structures of CMPs do not yet realistically mimic collagen fibers, rather, the term is used as a descriptor for nanofibers, microcages, hydrogels, liposomes etc. Also, some of the

CMPs discussed in chapter 4 formed stable triple-helices but have not yet been shown to form higher-order structures. These CMPs were included because they have unique synthetic schemes that are worthy of mention, such as synthetic routes to form heterotrimeric triple-helices or the use of a catechol-iron complex to raise the Tm of a

CMP by 22 °C, among others. With further research, CMPs may prove to be functional biomaterials, perhaps in lieu of collagen.

An important note to add is that different publications require different formatting for the rights and permissions to use pictures from the journal articles. The permissions have been adjusted to have some homogeneity but are not all in the exact same format.

1.2 Features of Collagen and Biosynthetic Process of Collagen Formation

Collagen is primarily composed of (2S)-proline (Pro, P), (2S,4R)-hydroxyproline

(Hyp, O), and glycine (Gly, G) (Table 2).1-5 The major repeat sequence of collagen is X-

Y-Gly, where X and Y can be any amino acid but are most frequently Pro in the X position and Hyp in the Y position.1-5 The most common triplet sequence in collagen is the Pro-Hyp-Gly (POG) sequence which accounts for approximately 10 percent of the sequence of collagen.2 Glycine is found in every third residue position in the collagen chain and makes a strong contribution to the formation of the collagen triple helix.2, 3 Gly is found on the inside of the triple-helix, whereas the amino acids in the X and Y positions are located on the outside of the triple helix and are therefore capable of intermolecular interactions. Substituting a different amino acid for a glycine in the third residue position results in a destabilization and loosening of the collagen triple helix.2, 5

This destabilization has pathological consequences including osteogenesis imperfecta

(OI).2, 5

Transcription and translation of genes that code for collagen, such as COL1A1

5 and COL1A2 among many others,6 yields a protocollagen strand that measures 0.8 nm by

300 nm long (Figure 1).2 The protocollagen strand undergoes enzymatic modification to

Amino Acid α1(I) chain α2(I) chain Glycine 345 346 Proline 127 108 Alanine 124 111 Hydroxyproline 114 99 Arginine 53 56 Glutamic Acid 52 46 Serine 37 35 Lysine 34 21 Aspartic Acid 33 24 Glutamine 27 24 Leucine 22 33 Threonine 17 20 Valine 17 34 Asparagine 13 23 Phenylalanine 13 15 Isoleucine 9 18 Methionine 7 4 Tyrosine 5 4 Hydroxylysine 4 9 Histidine 3 8

Table 2. The amino acid composition of type I collagen α1and α2 chains that form the type I collagen triple helix. The Pro-Hyp-Gly sequence (the corresponding amino acids are in bold) is the highest occurring sequence in the chains.1 Amino acids are organized by highest content in the α1(I) chain to lowest. form procollagen peptide strands which self-assemble to form a procollagen triple helix.

The enzymes involved in the process are lysyl hydroxylase, protein disulfide isomerase, prolyl 4-hydroxylase (P4H), and prolyl 3-hydroxylase (P3H).2, 3, 6 Lysyl hydroxylase oxidizes the lysine side-chains of the protocollagen strand. This oxidation forms hydroxylysyl pyridinoline and lysyl pyridinoline cross-links between protocollagen

6 strands and contributes to the formation of higher-ordered structures.2, 6 P4H and P3H modify proline residues in the X and Y position of the XYG sequence. P4H modifies a

Figure 1. Schematic of the biosynthesis of collagen. Genetic coding for collagen is transcribed to form a protocollagen strand. The protocollagen strand undergoes enzymatic modification to form a procollagen triple helix. Proteinases trim the N- and C- termini thus producing a tropocollagen triple helix which self-assembles into fibrils that cross-link to form fibers.2, 6 Reprinted with permission from Shoulder, MD, Raines, RT, Annual Review of Biochemistry, 2009, 78, 929-958 © 2009, Annual Reviews

Pro residue to (2S,4R)-4-hydroxyproline (Hyp) whereas P3H modifies Pro to (2S,3S)-3- hydroxyproline (3S-Hyp).6 Hyp is essential for the stability of the triple helix of collagen

7 and is frequently found in the Y position of the XYG sequence of type I collagen.2, 3 (3S)-

Hyp is frequently found in type IV collagen basement membranes in the X position of the

XYG sequence.2 A deficiency of P3H is associated with some forms of OI.2, 8, 9

A B

C D

Figure 2. TEM and AFM images of collagen displaying D-periodicity. A) TEM of collagen fibrils from human Achilles tendon stained with gold-palladium. Scale bar = 500 nm. B) TEM of type I collagen from calk-skin stained with phosphotungstic acid. Scale bar = 100 nm. C) AFM image of corneal collagen fibrils. Scale bar = 100 nm. D) AFM image of scleral collagen fibrils. Scale bar = 100nm. A,B) Adapted with permission from Electron Microscopy Reviews, 3/1, Chapman, JA, et al., The collagen fibril – a model system for studing the staining and fixtion of a protein, 143-182 © 1990 with permission from Elsevier. C, D) Adapted with permission from Japanese Journal of Ophthalmology, 46/5, Yamamto, S, Hitomi, J, et al., Observation of human corneal and scleral collagen fibrils by atomic force microscopy, 496-501 © 2002 with permission from Elsevier

After the enzymatic modification of the protocollagen strands is complete, the strands form a procollagen triple helix that is approximately 300 nm long.2 N- and C- proteinases cleave the termini to form a tropocollagen triple helix approximately 1-2 nm wide by < 300 nm long.2 The tropocollagen, henceforth referred to as a collagen 8 molecule, self-assembles into microfibrils that have a unique banding pattern that can be seen in two dimensions with a transmission electron microscope (TEM or SEM for scanning electron microscope) or an atomic force microscope (AFM) (Figure 2).2, 5, 10-12

This banding arises from the region of the microfibril where the tropocollagen strands gap and overlap with neighboring strands.2, 3, 5 This axial periodicity is referred to as the

D-period of collagen (Figure 1). The D-period of types I, II, and III collagen, which are the most abundant types of collagens, was measured to be 67 nm.2, 3, 5, 13 The length of the gap regions was 0.54D, and the length of the overlaps was 0.46D.2, 3, 5, 13 The few CMPs that display banding and periodicity did not mimic the 67 nm D-period and instead had shorter D-periodic lengths. Microfibril strands are cross-linked with each other through hydroxy-lysyl pyrodinoline and lysyl pyridinoline to form collagen fibers.2, 6 Collagen fibers can measure as long as a centimeter and 500 nm wide.2

The next two Chapters discuss how CMPs have been used to understand the physical properties of the triple helix of collagen and how specific amino acids contribute to the stability and formation of the triple helix. More specifically, Chapter two discusses the helical twist of collagen and CMPs, meaning how many amino acids constitute a twist in the triple helix. Chapter 3 explains how specific amino acids influence the stability of the triple helix by hydrogen bonding and stereoelectronic effects, among others. Chapter

4 discusses the higher-order structures and applications of CMPs. Many reviews have been written on the topic of CMPs but none provide as extensive of a review of higher- order CMP structures and applications of CMPs as this work.

CHAPTER 2

HELICAL TWIST OF THE TRIPLE HELIX OF COLLAGEN

To further understand the structure of the triple helix of collagen, X-ray crystallography of CMPs was used to determine how many residues constitute a turn in the triple helix. This is known as the helical twist (Figure 3). The CMP with a (POG)n

2, 5, 7 sequence, which closely mimics collagen, had a 72 helical twist. This means that seven X-ray scattering units (21 amino acids) form two turns in the triple helix.2, 5, 7, 14

The length of the helical twist is defined as the axial repeat. Triple-helices with a 72 helical twist have a 20 Å axial repeat. Triple-helices with a 103 helical twist have an axial repeat of 28.6 Å. Researchers in the 1950-1960 era found conflicting evidence that type I collagen has either a 72 or 103 helical twist, with 20 Å and 28.6 Å axial repeat units, respectively.15

16-18 In 1951, Cohen and Bear found type I collagen has a 72 helical twist.

Ramchandran and Kartha,19 on the other hand, proposed a coiled-coil three-stranded structure of type I collagen. In this model, each individual strand has a 101 helical twist

19 and the strands join together to form a 103 helical model. They proposed that the major

10 helix has a right-handed turn, whereas the individual strands are left-handed and the whole complex is held together by hydrogen bonds.19 Ramchandran also proposed that the residue sequence of collagen is (R-P-G)n, were R is any residue other than proline or hydroxyproline, G is glycine, and P is usually either proline or hydroxyproline but can be other residues as well.19

Kroner and Tabroff showed that only a minority of peptide sequences fit into the

RPG model that was proposed by Ramchandran and, instead, suggested that the major sequence in collagen is POG.20 Rich and Crick argued that there are stereochemical

Figure 3. A scheme of the helical twist and unit height parameters. In a 72 helical twist, there are 21 amino acids that constitute two turns of the helix and a unit height of 20 Å. Reprinted with permission from Biopolymers, 84/4, Okuyama, K, Wu, G, et al., Helical twists of collagen model peptides, 421-432 © 2006 John Wiley and Sons issues with the Ramchandran model that prevent hydrogen bonding. Based on these issues, the hybrid “Rich and Crick model"21 was developed, where POG is the major sequence, both proline and hydroxyproline can occupy the P and O positions, and there is

18, 21 a 103 helical twist as Ramchandran proposed.

By studying many CMPs it was found that some sequences, such as POG, display a 72 helical twist whereas other CMP sequences displayed a 103 helical twist. In 2012,

Okuyama15 was able to use crystallization to determine the full-cell structure analysis of

(GPO)9 at 1.45 Å resolution and showed that (GPO)9 forms a triple-helical structure, the individual collagen strands have a one-residue stagger, and GPO CMPs have a 72 helical twist.5, 18, 22-29 However, T3-785 is a CMP that mimics type III collagen, residues 785-796 and has an 11-residue guest sequence of which 9 residues were lacking imino acids.30, 31

30, 31 The 9-residues were flanked on both ends by POG sequences. T3-785 had a 72 helical twist in the POG regions and a 103 helical twist in the 9-residue sequence that lacked pyrrolidine rings.5, 14, 30-32 This research indicates that the helical twist changes along the triple helix of collagen and depends on the local sequences of amino acids.

To summarize, the relationship between the residue sequence of the peptide and the helical twist is complex. The helical twist has a sequence dependent variation, meaning the helical twist varies along the collagen and CMP chains as the residue sequence

5, 22, 33, 34, 13 changes. Imino acid-rich zones of a peptide have a tightly wound 72 helical twist, whereas imino acid-poor regions have a looser 103 helical twist. Both helical twists can exist on the same chain and arise from the variation of the amino acid residue sequence.2, 14, 22, 34, 13

CHAPTER 3

TRIPLE HELIX STABILITY AND AMINO ACID SEQUENCE

3.1 Introduction to Collagen Triple Helix Stability

CMPs have been used extensively to study how the amino acid content of a peptide influences triple helix stability. It was found that collagen triple helices are stabilized by pyrrolidine rings and Gly in every third residue.2, 3, 6, 7 The POG sequence stabilizes the triple helix of collagen with a network of interstrand hydrogen bonds and stereoelectronic effects. Charged amino acids can stabilize the triple helix through electrostatic interactions.3, 5 This section discussed how the sequence of amino acids stabilizes the triple helix.

3.2 Tertiary Amides

Researchers tested if the stability of the triple helix is dependent on the presence of proline and proline derivatives or if the stability arises from tertiary amides. N- isobutylglycine (Nleu), N-methylalanine (meAla), and Ala replaced Pro and Hyp of a

POG repeat sequence.35, 36, 37 Gly-Nleu-Pro sequences were found to have a higher stability than Gly-Pro-Nleu sequences.35, 37 These results indicated that in the

CMP Tm (°C) Reference (Gly-Pro-Nleu)9 39 35 (POG)3(POG)(POG)3 36 36 (Pro-Pro-Gly)10 35 35 (POG)3(PPG)(POG)3 30.5 36 (POG)4(AOG)(POG)5 26.1 36 (POG)3(methylAla-Hyp-Gly)(POG)3 17.5 36 (POG)3(Pro-methylAla-Gly)(POG)3 21.7 36

Table 3. The stability effects of tertiary amides in comparison to POG and PPG sequences. Tertiary amides other than Nleu tend to have a destabilizing effect on the Tm of CMP triple-helices. presence of Nleu, a Pro in the Y position is stabilizing, which goes against the normal trend. Although Nleu showed to have a stabilizing effect compared to a Pro-Pro-Gly

CMP, meAla and Ala substitutions had destabilizing effects on the triple helix (Table

3).35, 36 This research indicates that the proline rings provide stability to the triple helix and the stability arises from other properties than Pro and Hyp being tertiary amides.36

3.3 Stereoelectronic Effects

The ring puckering of the pyrrolidine rings of Pro and Hyp have a stereoelectronic effect that stabilizes the triple helix. Stable triple helices generally by having a Cϒ-endo ring puckering in the X position and a Cϒ-exo ring puckering in the Y position (Figure

4).2, 5 The ring puckering of the pyrrolidine rings orients the main chain dihedral angles of procollagen strands into the positions required to form the triple helix.38, 39

Figure 4. Cϒ-endo and Cϒ-exo ring puckering of the pyrrolidine ring residues. Proline or proline derivatives with Cϒ-endo ring puckering in the X position and Cϒ-exo ring puckering in the Y position provide stability to the collagen triple helix through stereoelectronic effects. These stereoelectronic effects orient the main chain dihedral angles of the collagen chain to form a triple helix.

The CMP (Hyp-Hyp-Gly)10 had triple helix with a high stability of 65°C, in

2 contrast to (Pro-Pro-Gly)10 which had a lower stability of 31°C. This indicates that the

Cϒ-exo ring puckering in the Y position has a strong impact on the stability of the triple helix. (3S)-Hyp had a Cϒ-endo ring puckering and provided stability when in the X

15 position of the (XYG) sequence of CMPs but (3S)-Hyp decreased the Tm of the CMP when in the Y position.40 This is to be expected as (3S)-Hyp has a Cϒ-endo ring puckering that is favorable in the X position but not in the Y position.

To further show that stereoelectronic effects play a major role in stabilizing the collagen triple helix, fluorinated prolines were incorporated into CMPs. Fluorine is

CMP sequence Tm (°C)

(Pro-Flp-Gly)10 91

(POG)10 61-69

(Hyp-Hyp-Gly)10 65

(flp-Pro-Gly)10 58

(PPG)10 31-41

(POG)3-(3S-Hyp)-Hyp-Gly-(POG)3 32.7

(POG)3-Pro-Pro-Gly-(POG)3 30.5

(flp-Flp-Gly)10 30

(POG)3-Pro-(3S-Hyp)-Gly-(POG)3 21

(POG)7 36

(Flp-Pro-Gly)10 No helix

(Pro-(3S-Hyp)-Gly)10 No helix

Table 4. Stereoelectronic effects contribute to the stability of the triple helix as shown by the use of fluorinated derivatives of proline. Tm values from Ref. 2. incapable of forming hydrogen bonds and therefore the fluorinated CMP triple helix can only be stabilized by stereoelectronic effects. Using (4R)-fluoroproline (Flp) and (4S)- fluoroproline (flp) as substitutions for Pro and Hyp in the POG sequence of CMPs,

2 Raines showed that (Pro-Flp-Gly)10 has a high Tm of 91°C (Table 4). This Tm is higher than that of a (POG)10 CMP thus showing that stereoelectronics play a major role in triple helix stability independent of hydrogen bonding. It has also been shown that (flp-Flp-

Gly)7 and (Pro-Pro-Gly)7 CMPs bind to collagen in vitro and ex vivo and that these peptides are non-toxic to human fibroblast cells.41

3.4 Hydrogen Bonding

N-H(Gly) forms interstrand hydrogen bonds with the carboxyl group of the Pro in

2, 3, 7 the X position (N–H(Gly)--C=O(Pro)) on a separate strand in the triple helix (Figure 5).

This hydrogen bond was proposed by Rich and Crick in 1955 and was later confirmed by

Bella et al. in 1994 using 1.9 Å resolution X-ray crystallography.7 The triple helices of

Figure 5. A schematic diagram of a one-residue stagger and interchain hydrogen bonding between three strands (dashed line) that enables triple helix formation in procollagen and (POG)n CMPs. collagen are also stabilized by interchain hydrogen bonds between Gly and the residue in

14, 28, 33, 42 the X position other than Pro (N–H(Gly)--C=O(X)). There may also be a water- mediated hydrogen bond between Gly and the residue in the X position (N-H(Gly)---H2O--

14 -O=C(X)). It has also been shown that when Gly is substituted with Ala there is a loss of interchain hydrogen bonding and is replaced by water-bridge-mediated hydrogen bonding

28, 42-44 between alanine and proline in neighboring chains (N–H(Ala)--H2O--C=O(X)).

(4S)-acetamidoproline (Acp) and (4S)-formamidoproline (Fmp), which have a

Cϒ-endo ring puckering, were used as substitutions for Pro and Hyp in the POG sequence.45 It was found that a Cϒ-endo ring puckering in the Y position could still form a stable triple helix because the hydrogen bonding is sufficient to stabilize the triple helix

(Table 5). This indicates that hydrogen bonding has a strong contribution to the stabilization of the triple helix.45

CMP Tm (°C)

(POG)3-(Pro-Acp-Gly)-(POG)3 40

(POG)3-(Pro-Fmp-Gly)-(POG)3 39

(POG)7 43

(POG)3-(PPG)-(POG)3 40

Table 5. Acp and Fmp were used to show that even though a residue may not have the appropriate ring puckering for the position it is in, the hydrogen bonding network can provide stability to the triple helix. Tm values from Ref. 45

3.5 Electrostatic Interactions

In addition to stereoelectronic interactions and hydrogen bonding, electrostatic interactions can also stabilize the triple helix of collagen. Oppositely charged amino acids

18 in the sequence of a CMP or collagen form interstrand electrostatic interactions that provide stability to the triple helix. 15 to 20% of the sequence of collagen is composed of charged amino acids.46 Charged amino acids found in collagen include Asp (D), Lys (K),

Arg (R), and Glu (E).46, 47 To investigate the role of electrostatic interactions, a CMP with the sequence (Gly-Pro-Hyp)3-Gly-X-Y-Gly-X’-Y’-(Gly-Pro-Hyp)4 was designed. Lys,

Arg, Glu, and Leu, were studied in the X, X’, Y, and Y’ positions.48 They found that

48 GPKGEO and GPKGDO guest sequences have a stability close to a (GPO)9 sequence.

These CMPs containing charged amino acids showed that electrostatic interactions can stabilize the triple helix of collagen and CMPs. Electrostatic interactions have also been incorporated in CMPs to form higher-order structures such as fibers with

D-periodic spacing and hydrogels. This will be discussed in Chapter 4.

3.6 The Role of Glycine in Triple Helix Stability

Glycine is required in the third position in order to form the triple helix.1-5, 49 OI is the result of a missense mutation where a single base replaces glycine with a different amino acid49, 50-53 and causes a defective mineralization of type I collagen in bones.54, 55

OI is commonly referred to as brittle bone disease because the bones are fragile and susceptible to fracture. Patients with OI tend to live shorter lives, have a blue scleral hue

55 in the eyes, and suffer from hearing loss, among other problems. The Tm of a (GPO)4-

56 APO-(GPO)4 CMP (Figure 6), which substitutes Ala for Gly, has a low Tm of 28 °C.

Many different kinds of OI have been identified and the lethality of the particular type of OI is dependent on the amino acid replacing glycine as well as the location of the substitution in the chain.49, 53, 54 CMPs with various Gly substitutions have a discontinuous triple helix, a lower melting temperature and a change in their helical twist,

28, 49, 57, 58 thus destabilizing the triple helix. The destabilization of Tm depends on the residue used as the substituent and on the location of the substitution relative to the N- and C- termini. A substitution near the N-terminal can be less lethal than a substitution near the C-terminal.42, 53 The sequence of residues neighboring the mutation site as well as on the chain that contains the mutation also affects the destabilization of the triple helix.49, 57, 58 For example, Brodsky and coworkers showed that all Gly→Asp substitutions in the α1 chain have a lethal effect. In contrast, a Gly→Cys substitution in the α1 chain is non-lethal, if it occurs in a specific location near the N-terminus, but it can be lethal in other locations on the same chain.49

Figure 6. CMP design with a Gly→Ala substitution.49 This CMP mimics the defective collagen found in patients suffering from OI.

The sequence, Cys-Gly-Lys-Hyp-(Gly-Pro-Hyp)2-Gly-Glu-Hyp-(Gly-Pro-Hyp)2-

Gly-Arg-Hyp-X-Pro-Hyp-Gly-Pro-Hyp-Gly-Asp-Hyp-(Gly-Pro-Hyp)5, was studied as an

OI CMP.59 This sequence was chosen as it is known to mineralize with hydroxyapatite, the mineral in bones. In this OI CMP, X = Gly, Ala, Val, or Asp and these OI CMPs were studied as films that were mineralized with hydroxyapatite. Ala, Val, and Asp were chosen because these substitutions of Gly known causes OI and these amino acids were also chosen for their hydrophilicity and stereo-hindrance properties. Ala is a small and hydrophobic residue, Asp is bulky and hydrophilic, and Val is bulky and hydrophobic. OI bones have lower Ca/P molar ratios than healthy bones and it was found that Ala had a lower Ca/P molar ratio than Val and Asp.59 The mineral thickness of the OI CMPs decreased when X = Val or Asp and there is no difference in mineral thickness on the OI

CMP films when X = Gly or Ala.34, 59

In order to form triple helices, all the amino acids in the strand must be in a trans position. Researchers replaced Gly in a POG CMP with a trans-locked alkene isostere

(tlAI).60 This design did not inhibit any interstrand hydrogen bonding in the CMP.

Interestingly, although the hydrogen bonds remained intact and all the residues were

60 locked in the trans conformation, the tlAI CMP had a low Tm of 28.3 °C. This research further illustrates the important role of Gly in stabilizing the triple helix.

3.7 Conclusion of Triple Helix Stabilizing Factors

In summary, the amino acid sequence impacts the network of interchain hydrogen

21 bonding and stereoelectronic effects from the ring puckering of Pro and Pro derivatives also influence the stability of the triple helix of collagen. Electrostatic interactions between charged amino acid residues can also stabilize the triple helices. Substituting glycine in the third position of the XYG sequence with another amino acid destabilizes the triple helix and has pathological consequences such as OI. These findings help to explain the self-assembly process of the triple helix of collagen. Understanding the assembly of the triple helix of collagen is essential to understand how collagen self- assembles into higher-order structures to form fibers. The following section focuses on strategies for designing CMPs to have triple helices, higher-ordered structures, and biological applications.

CHAPTER 4

HIGHER-ORDER CMP STRUCTURES

4.1 Intro to Higher-Order Structure and Applications of CMPs

Collagen fibrils self-assemble into larger collagen fibers that have a 67 nm D- periodic spacing, ~500 nm diameter, and a length on the order of a centimeter. In this section, numerous methods used to design CMPs capable of forming higher-order structures and their potential applications are reviewed. The techniques used to create these CMPs include the use of cysteine knots, electrostatic interactions and aromatic interactions, among others. It is important to note that the term ‘higher-order structures’ is used quite liberally and generally refers to the formation of nanofibers and hydrogels, although also includes other structures such as liposomes, nano-cages, films, etc. CMPs are far away from mimicking collagen fibers and although certain features of collagen have been reproduced, they are not on the same order as collagen. For example, type I collagen has a 67 nm D-periodicity and there are CMPs that have this feature, but the D- periodicity of these CMPs is tens of nanometers smaller than that of collagen.61, 62, 63

This section also focuses on applications of CMPs, however, for the most part,

CMPs are are not yet robust and functional biomaterials. Many CMPs have been shown to be capable of cell adhesion and viability, but these are only preliminary studies and have yet to show cellular functionalities on CMPs. There are CMPs that do have some functionality, but overall CMPs need further research to prove as viable alternatives to collagen. Finally, there are CMPs that are discussed here that have not yet been shown to form higher-order structures but are worthy of note as they have unique synthetic schemes, such as the synthesis of heterotrimeric CMPs, or because they possess some other unique feature, such as nanodiamond-conjugated CMPs. This section provides a comprehensive review of higher-order structures and applications of CMPs, of which many have not been discussed in previous reviews. Also, most reviews have only focused on certain subsections of this topic, whereas this work encompasses a larger scope and provides a fair amount of detail.

4.2 Pro-Hyp-Gly CMPs

The (POG)10 CMP promotes self-assembly and forms branched fibrillar structures with irregular branching of the fibers (Figure 7A).14, 64 Also, the fibers are found as aggregates and not as solitary strands.64 The POG CMP is similar to collagen in a few ways. Fibers form at neutral pH and with increasing temperature.64 Sugars inhibit the

64 fiber formation of collagen and the POG CMP. Also, both the (POG)10 CMP and collagen undergo fibrillogenesis in a nucleation-growth mechanism.64 However, Collagen forms highly organized fibers whereas the POG CMP had the irregular branching and aggregation.

Figure 7. TEM of (POG)10 and polyPOG. A) (POG)10 with branched extensive branching 64 65 of the fibers. B) (POG)10 aggregates of nanofibers. C) poly-POG fibers displaying 65 higher dispersion of fibers than (POG)10. A) Reprinted with permission from The Journal of Biological Chemistry, 281/44, Kar, K, Amin, P, et al., Self-association of collagen triple helic peptides into higher order structures, 33283-33290 © 2006 American Society for Biochemistry and Molecular Biology B, C) Adapted with permission from Biopolymers, 79/3, Kishimoto, T, Morihara, Y, et al., Synthesis of poly (Pro–Hyp–Gly)n by direct polycondensation of (Pro–Hyp–Gly)n, where n= 1, 5, and 10, and stability of the triple‐helical structure, 163-172 © 2005 John Wiley and Sons

In another experiment, POG was polymerized into a high molecular weight (MW

= 10,000) POG polypeptide using a polyamide-condensation reaction and compared it to

65 a (POG)10 CMP. Both, the polyPOG and (POG)10 formed fibers had a diameter of

65 roughly 10 nm and were found as aggregates of fibers. The aggregates of the POG)10 fibers were roughly 5.5 μm in diameter (Figure 7B), whereas the polyPOG fibers were less aggregated and more dispersed (Figure 7c).65

4.3 Pro-Pro-Gly CMPs

64, 66 The (PPG)10 CMP does not form higher-ordered assemblies. PPG supramolecular structures are desirable because E. coli expression can be used to mass produce

Figure 8. TEM of nanofibers from a CMP with the sequence (GPP)3GPR-GEKGER- 72 GPR(GPP)3-GPCCG at different scales. A, B) Scale bar = 0.5 μm and C, D) Scale bar = 1μm. Reprinted with permission from Biomacromolecules, 32/7, Krishna, O. D., and Kiick, K. L., Integrin-mediated adhesion and proliferation of human MSCs elicited by a hydroxyproline-lacking, collagen-like peptide, 6412-6424 © 2009 American Chemical Society recombinant proline-rich peptides. However, E. coli lack the enzymes required to produce hydroxyproline. It is, therefore, important to investigate whether PPG CMPs are capable of forming higher-ordered structures and have practical applications. Using

CMPs with PPG sequences, researchers designed triple-helical peptides lacking hydroxyproline.67-71

A PPG CMP was designed with the sequence (GPP)3GPRGEKGERGPR(GPP)3-

GPCCG.72, 73 This CMP incorporated a type III collagen cysteine (C) knot at the C- terminus and GPR sequences to promote triple helix formation. The GEKGER sequence 26 in the CMP added stability to the CMP triple helix through electrostatic interactions, is known to attract integrins, and is found abundantly in many collagens. This CMP formed

Figure 9. A) Schematic of PPG CMP F877. The underlined G at position 901 was substituted with Ser in the G901S CMP. B) TEM of F877 nanofibers. Scale bar = 40 nm. C) TEM of G901S fibers. Scale bar = 40nm. Adapted with permission from Angewandte Chemie International Edition, 46/18, Bai, H, Xu, K, et al., Fabrication of Au nanowires of uniform length and diameter using a monodisperse and rigid biomolecular template: collagen‐like triple helix, 3383-3386 © 2007 John Wiley and Sons nanorods in addition to 6 μm fibrils with a 130 nm width, and modulated human mesenchymal stem cell adhesion and migratory responses.72, 73

E. coli was used to express two PPG CMPs, F877 and G901S, that served as a template for the formation of gold as well as ZnO nanowires that are 4 nm wide and 40 nm long.68, 74 The F877 CMP mimicked human type I collagen, residues α1(887-939) and contained PPG units for stability, cysteine residues for interchain disulfide bridges.68

F877 had a Tm of 42 °C and mineralized with gold after four days of incubating in

68 trimethylphosphinegold chloride (AuPMe3Cl). In another experiment, the F877 CMP

27 also mineralized with ZnO. The length of these gold and ZnO nanowires could be tailored by the number of amino acid residues in the sequence.68, 74 The G901S CMP was

Figure 10. A) TEM of the cubes assembled by streptavidin-labelled gold nanoparticles and a biotinylated F877 CMP. Scale bar = 1μm. B) TEM of the hexagonal structures that assembled when the diameter of the gold nanoparticles was raised to 30 nm. Scale bar = 1μm. Adapted with permission from Angewandte Chemie International Edition, 49/45, Kaur, P, Maeda, Y, et al., Three‐dimensional directed self‐assembly of peptide nanowires into micrometer‐sized crystalline cubes with nanoparticle joints, 8375-8378 © 2010 John Wiley and Sons designed with the same sequence as the F877 CMP, however the Gly residue at position

901 was substituted with Ser. The G901S CMP had a lower Tm of 30 °C and did not mineralize with gold or ZnO.68, 74

Another CMP that is similar to the F877 CMP was produced by E. coli but designed with an additional 15-residue sequence at the N-terminus, which functions as a biotin acceptor peptide.75 Gold nanoparticles (diameter = 10 nm) were coated with streptavidin, which bound to the CMP via streptavidin-biotin interactions. This CMP formed 1-2 μm cubes with peptide-nanowires as frames and gold nanoparticles as the joints of the cube.75 The cubes were highly uniform in both dimension and shape. By reducing the concentration of peptide and gold nanoparticles, researchers obtained smaller cubes approximately 100-200 nm in length.75 Reducing the pH to slightly below 28 neutral yield cubes 20-80 nm long.75 By using gold nanoparticles with a higher diameter of 30nm, the complex formed micron-sized hexagonal structures.75

Figure 11. A) Schematic of the B877B CMP. B) Schematic of the process of forming microcapsules on the octane/water interface. Streptavidin was used to lock the structure of the microcapsules. C) Illustration of the assembled structure of the microcapsules. D) Fluorescence micrograph of the microcapsules. Streptavidin-stabilized microcapsules have a higher contrast. E) TEM of B877B microcapsules when streptavidin was bound to quantum dots. The inset image is a magnified image of the QD-streptavidin-B877B microcapsules. Adapted with permission from Small, 8/9, Maeda, Y, Wei, Z, Matsui, H, Biomimetic assembly of proteins into microcapsules on oil‐in‐water droplets with structural reinforcement via biomolecular‐recognition‐based cross‐linking of surface peptides, 1341-1344 © 2012 John Wiley and Sons

In another experiment, instead of gold nanoparticles, quantum dots were bound to the peptide and this formed free-standing films with a Young’s modulus of ~20 GPa.76 In another experiment, the F877 CMP (B877B) was designed with biotin at both the N- and

C-termini. The peptide was vigorously shaken in a mixture of water and octane (10:1 29 volume ratio) and formed microcapsules on the oil-in-water droplets.77 The microcapsule structures were then crosslinked by streptavidin to add stability by locking the structure of the microcapsules (Figure 2A-D). The microcapsules ranged in size, from 4 – 170

μm.77 Microcapsules were also formed when streptavidin was also bound to quantum dots (QD) and bound with B877B (Figure 2E).77

Expanding on the work involving biotinylated CMPs, researchers also designed biotinylated POG and DOG CMPs.78 Within seconds of adding avidin to the solution, the peptides self-assembled into translucent, free-floating films. The films were square, roughly 1mm in length, and had a relatively uniform thickness of ~9 μm. Solutions with higher concentrations of avidin formed thicker films.78 The researchers were hoping to yield fibers and this was an unexpected result.78

4.4 CMP Amphiphiles

Peptide amphiphiles (PAs) contain a peptide head-group and a hydrophobic tail.

PAs have been shown to potentially possess numerous biological applications such as drug delivery and tissue regeneration and also form a wide range of structures.81 PAs are also capable of eliciting specific cellular and enzymatic responses using specific peptide sequences.79, 80, 81

Fields et al. designed CMPAs with long-chain dialkyl ester lipid tails and

30 monoalkyl chain tails with a head group which self-assembled into triple helices and spherical micelles. The head group was based on the type IV collagen α1 chain, residues

α1(IV)531-543, α1(IV)1263-1277 (IV-H1) (Figure 6), α1(IV)119-122, and α1(IV)382-

393.82-87 The CMPAs had dialkyl tails of various lengths next to a Glu linker followed by a (CH2)2 spacer and ending with a CMP head group. The dialkyl ester compounds were found to stabilize the triple helix for carbon chain lengths of 12-18 carbons.82-87 The stability of the triple helix increased as monoalkyl chain lengths increased from a six carbon tail that had a Tm of 42.2 °C to a tail with sixteen carbons, which had a Tm of 69.8

88 °C.

A CMPA mimicking type IV collagen, residues α1(IV)1263-1277, with the sequence Gly-Val-Lys-Gly-Asp-Lys-Gly-Asn-Pro-Gly-Trp-Pro-Gly-Ala-Pro, were found to bind with CD44/chondroitin sulfate proteoglycan receptors.90 These receptors are overexpressed in metastatic melanoma cancerous cells. Liposomes composed of

Figure 12. Structure of the IV-H1 CMPA83 where the 15-residue guest sequence mimics collagen sequence α1(IV)1263-1277. Melanoma cells were capable of adhesion and spreading on surfaces coated with the IV-H1 CMP.89 distearoyl phosphatidylcholine, distearoyl phosphatidylglycerol, and cholesterol were designed to interact and bind with the CMPA.79 Rhodamine, a cellular fluorophore was encapsulated in the liposome. The hydrophobic tail of the CMPA inserts itself into the

31 lipid bilayer of the liposome and the polar head group remains on the surface of the

90 liposome. The CMPA bound the liposome with the CD44/chondroitin sulfate

Figure 13. A) Schematic of α1(IV)1263-1277 CMPA sequence and the structure of its triple helix. B) The assembled liposome loaded with a fluorophore. C) The CMPA binds with the liposome by inserting the hydrophobic tail onto the bilayer of the liposome. D) The CMPA epitope segment binds with the CD44 receptors of a melanoma cell and releases the cargo from the liposome. Reprinted with permission from Journal of the American Chemical Society, 129/16, Rezler, EM, Khan, DR, et al., Targeted drug delivery utilizing protein-like molecular architecture, 4961-4972 © 2007 American Chemical Society proteoglycan receptors, the liposome was then brought into the tumor cell, and the encapsulated rhodamine was released inside the cell.79 In vivo, an α1(IV)1263-1277

CMPA/liposome loaded with doxorubicin, a medicine used in cancer chemotherapy, reduced the size of tumors to 60% of the untreated control in vivo.90 This technique enables one to control and target drug delivery by tailoring specific sequences than can

32 bind with specific receptors on the cell of interest.

Another liposome was designed, composed 70% of 1-palmotoyl-2-oleoyl-sn- glycero-3-phosphocholine and 30% CMPA with the collagen mimetic sequence: H2N-

80 GPQGIAGQR(GPO)4GG-COOH. This sequence provided the cleavage site for the

Figure 14. A, B) TEM of CMPA container a GFOGER integrin-binding guest sequence. These fibers have a ~16nm diameter. A) Scale bar = 200nm. B) Scale bar = 50 nm. C) The CMPA was able to form a nanofibrous hydrogel. D) The vial containing the hydrogel was inverted to show that the hydrogel is self-supporting.96 Adapted with permission from ACS Nano, 5/10, Luo, J, and Tong, YW, Self-assembly of collagen-mimetic peptide amphiphiles into biofunctional nanofiber, 7739-7747 © 2011 American Chemical Society matrix-metalloproteinase enzyme, MMP-9. The MMP-9 enzyme degrades the extracellular matrix and indicates the progression and metastasis of tumors in humans including breast, colorectal, lung, and prostate tumors among others.80, 91-94 This enzyme cleaved the CMPAs in the liposome, which destabilized the lipid domains of the liposome. The destabilization of the liposome caused the release of carboxyfluorescein, which was encapsulated in the liposome.80 This technique could be used to image tumors with fluorescent particles or could potentially be used as a drug delivery system.

Microwave synthesis was used to create a collagen mimetic lipopeptide with the

95 sequence CH3(CH2)16CONH-GPQGIAGQR(GPO)4GG. This sequence is known from earlier work80 to contain the sequence for the MMP-9 cleavage site. This new synthetic method was able to yield triple-helices and the synthesis is faster than traditional solid-

33 phase peptide synthesis.95

Another CMPA was developed and was composed of four segments: a lipophilic tail, a β- sheet-forming segment, a lysine spacer, and an epitope segment.96 The CMP sequence in the epitope segment was (GPO)3(GFOGER)(GPO)3 and incorporated the GFOGER sequence (Gly-Phe-Hyp-Gly-Glu-Arg) which is known to bind to numerous

Figure 15. A) An overall schematic showing the PA self-assembled into a nanofiber and then underwent the self-templating process and was pulled to form a self-tamplated pattern on a surface. B) TEM image of a nanofiber. Scale bar = 50 nm. C) AFM image of nanofibers. Scale bar = 1 μm. D) Self-tamplating yielded a twist plywood pattern with vertical periodic spacing. Top - scale bar = 50 μm. Bottom – scale bar = 20 μm. E) Crimp pattern achieved from the self-templating process with a periodic horizontal pattern. Top – scale bar = 40 μm. Bottom – scale bar = 20 μm. F) Scheme showing MC3T3-E1 cells and HA were seeded ontp the pattern scaffold. G) Bright field and fluorescent microscopy images of MC3T3-E1 preosteoblast cells that were cultured for 5 days on a self-templated surface aligned with the grooves (scale bar = 100 μm, actin in green, nuclei in blue). Adapted with permission from Nano Letter, 15/10, Jin, HE, Jang, J, et al., Biomimetic self-templated hierarchical structures of collagen-like peptide amphiphiles, 7138-7145 © 2015 American Chemical Society integrins. These CMPAs formed nanofibers with a diameter of 16 nm and micrometer length together with hydrogels capable of human liver carcinoma cell (HepG2) adhesion and spreading (Figure 14).96

Chung and coworker designed a CMPA with a 12-residue sequence,

NPYHPTIPQSVH, known to be a binding site for hydroxyapatite.97 The mimetic sequence was followed by a spacer region, then a cross-linking segment, and ended with a hydrophobic tail.81 This CMPA assembled into nanofibers with a radius of ~17nm. The self-templating process98 was used to deposit the CMPA onto a substrate. The substrate was immersed in solution and then pulled vertically leading to a deposition of CMPA in a hierarchical pattern on the substrate.81 When pulling, there are competing interfacial forces, such as friction and surface tension, at the air-liquid-solid interface. Two distinct hierarchical patterns that formed were twisted-plywood and crimped filament structures.81 The twisted plywood structure was composed of a striped pattern and crimped filament was described as periodic zigzag morphologies with a ridge-groove structure. To compare to collagen, in tendon tissues, collagen forms a crimp pattern and in decalcified bone, collagen displays a twisted plywood structure.81 MC3T3-E1 preosteoblast cells on the crimp pattern grew parallel to the ridge/groove direction, On the twisted-plywood surface, the cells grew in a uniform direction.81 The HA CMPA was

2+ 3- also mineralized with a solution of Ca , PO4 , and polyaspartate. The crystal deposition on the ridged/grooved template followed the pattern and did not disrupt the grooved pattern.81

4.5 2+1 Strand Click Synthesis

A heterotrimeric POG CMP was developed using a technique called 2+1 strand click synthesis to synthesize heterotrimeric triple-helices.99 The “2” refers to the fact that two chains were linked by means of a C-terminal lysine with a 1,5-diaminopentane

Figure 16. Schematic diagram of Byrne et al.99 for 2+1 strand click synthetic chemistry. Reprinted with permission from Chemical Communications, 47/9, Byrne, C, McEwan, PA, et al., End-stapled homo and hetero collagen triple helices: a click chemistry approach, 2589-2591 © 2010 with permission of The Royal Society of Chemistry spacer.99 The “+ 1” denotes that a third strand containing (2S)-propargylglycinamide

(Pra) “clicks” with a 6-azidolysine residue located at the C- terminus of the dual strand.99

This strategy was initially used to design heterotrimeric POG CMPs with integrin- binding guest sequences (Figure 7).99

This technique was further developed to design three heterotrimeric POG CMPs

36 with an α1(I-III)769-783 guest sequence and a phosphinate enzyme inhibiting sequence.100 These CMPs have not yet been shown to form higher-ordered structures but they do form stable triple helices and they were found to inhibit the activity of certain matrix metalloproteinase enzymes.100

4.6 Cysteine Bridges

Type III collagen contains two cysteine knots at the C-terminus that form three

Figure 17. Scheme of the triple helix of a heterotrimeric POG CMP.102 Cysteine knots (green) gave researchers the ability to add stability to the triple helix. The cysteine knots also enabled the ability to form a heterotrimeric CMP. disulfide bonds between the three chains of its triple helix.47, 101 Cysteine residues have been incorporated into CMPs in order to mimic type III collagen to determine their ability to form cysteine knots that stabilize the triple helix and to form higher-ordered structures.

To date, researchers were able to design materials with cysteine knots capable of forming fibrils, hydrogels, and free floating films.

Cysteine knots were incorporated into (POG)5 CMPs to test how the disulfide

47 bridges affect the stability of the triple helix. A simple (POG)5 CMP is less stable

Figure 18. Schematic for the assembly of a hydrogel from the self-assembly of staggered triple helices. Reprinted with permission from Biopolymers Peptide Science, 90/6, Yamazaki, CM, Asada, S, et al., Artificial collagen gels via self‐assembly of de novo designed peptides, 816-823 © 2008 John Wiley and Sons compared to a (POG)5 CMP with cysteine knots. The former has a Tm of 28 °C while the

47 latter has a Tm of 68 °C. A POG heterotrimeric triple helix was designed and stabilized through the use of cysteine knots. (Figure 17).102 These CMPs produced collagen-like fibrils 20 - 120 nm in length and 0.5 – 1 nm wide.102 Collagen fibrils have a similar diameter but are significantly longer in length.102

Another study made use of cysteine knots to form stable triple-helices, in which

24-mer POG CMPs were linked by disulfide bonds. These CMPs had a 12- or 13-residue stagger and formed triple helices.103 For comparison, the three strands of a collagen triple

Figure 19. The design schematic of the POG based CMP with either GFOGER or GPOGER integrin-binding sequences.105 The triple helices self-assemble into nanofibers and were capable of cell adhesion. Reprinted with permission from Biopolymers, 31/7, Yamazaki, CM, Kadoya, Y, et al., A collagen-mimetic triple-helical supramolecule that evokes integrin-dependent cell responses, 1925-1934 © 2010 Elsevier

helix are staggered by one residue. To explore these CMPs further, 27-mer POG peptides also linked by Cys bridges were developed. These CPMs had 12-, 15-, and 18-residue staggering. Arg was attached to the C-termini of the peptides to generate hydrophilicity and stability.104 These CMPs self-assembled into thermally reversible hydrogels while the control POG CMP lacking Arg did not form hydrogels.104 Similar POG CMPs with cysteine knots were designed and additionally contained the GFOGER and GPOGER integrin binding sequences (Figure 19).105 These CMPs formed rod-shaped structures less than a micron in length with 0.5-1 nm diameters and were capable of human dermal fibroblast cell adhesion.105 39

4.7 Aromatic Interactions

The use of aromatic interactions to form triple helices and higher-ordered structures of CMPs is a novel technique. Aromatic interactions are non-covalent end-to-

Figure 20. A) Chemical structure of the CMP from Ref. 106. B) Image showing the interactions of aromatic interactions of peptides in the triple helix. C) The CMP is capable of aggregating platelets similarly to collagen. D) TEM of CMP nanofibers. E) TEM of a cross-section of a murine blood vessel. Adapted with permission from the Journal of the American Chemical Society, 129/8, Cejas, MA, Kinney, WA, et al., Collagen-related peptides: self-assembly of short, single strands into a functional biomaterial of micrometer scale, 2202-2203 © 2007 American Chemical Society end interactions and include π-π stacking, CH-π interactions, as well as cation-π interactions. These can be incorporated into CMPs and may enable the formation of triple helices as well as fibrils, fibers, and nanowire templates.

To show that aromatic interactions can be used to form fibers, Maryanoff and coworkers designed a single-stranded CMP using a POG peptide with phenylalanine and pentaflourophenylalanine end groups at the N- and C-termini (Figure 20A).106 This CMP self-assembles into a bioactive material. Phenylalanine has an electron-rich benzyl ring, 40 whereas the hexafluorobenzene in pentafluorophenylalanine, is an electron-poor benzyl ring.106 The design utilized the principles of aromatic stacking and ordered hydrophobic

Figure 21. Chemical structure of the CMP containing phenylalanine and pentaflourophenyl termini groups and schematic to yield gold-labelled nanowires. B) TEM of CMP fibers before electroless silver plating. C) TEM of CMP fibers after electroless silver plating. D) TEM of CMP coated with silver nanoparticles after one deposition and one hour. Adapted with permission from the Journal of Materials Chemistry, 18/32, Gottlieb, D, Morin, SA, et al., Self-assembled collagen-like peptide fibers as templates for metallic nanowires, 3865-3870 © 2008 with permission of The Royal Society of Chemistry interactions to produce fibers with lengths om the micrometer-scale and diameters ~0.26

μm. The fibers were reported to resemble murine aortic tissue fibers and induce the aggregation of platelets.106, 107

Extending on the work of Maryanoff,106 a triple-helical CMP was designed using

π- π stacking between phenylalanine and pentaflourophenyl termini groups (Figure

21A).108 However, this CMP was incorporated with a Hyp→Lys substitution in order to bind with gold nanoparticles.108 This CMP self-assembled into micrometer length fibers and could be made into silver nanowires incorporated with gold nanoparticles (Figure

21B-D).108 Nanowires have applications in nanosensors and microfluidic devices among others. Lithography can be used to design nanowires, however, it is very costly.

Therefore, researchers try to design peptide based self-assembling nanowires.

CH-π interactions occur between aromatic phenylalanine and tyrosine residues with imino acid residues on separate triple helices.109 One of the results from this experiment was that the rate of triple-helix formation was accelerated in comparison to a

POG peptide.109 These peptides formed fibers 20-40nm long and induced platelet aggregation.109

A (POG)3-capped, Pro-Y-Gly-X-Hyp-Gly sequence was researched, where Y was an aromatic residue and X was a positively charged cationic amino acid.110 This CMP was used to show that cation-π interactions within the triple helix have a stabilizing effect.110 In another POG CMP, arginine was located at the N-terminus and phenylalanine at the C-terminus of the CMP.111 This design resulted in a favorable cation-π interaction between Arg and phenylalanine that resulted in a head-to-tail assembly of the triple helix and resulting in micrometer scale fibrils.111

4.8 Metal-Induced Self-Assembly

Researchers have used metal-induced self-assembly to design CMPs with triple helices and capable of forming higher-ordered structures that have biological functions

Figure 22. A) Chemical structure of the KByp-containing CMP from Ref 112. B) The peptide strands self-assemble into a triple helix. C) Upon the addition of Fe(II), the triple- helices grow in the radial direction to form fibers. D) TEM image of the fibers. Adapted with permission from the Journal of the American Chemical Society, 130/38, Przybyla, DE, and Chmielewski, J, Metal-triggered radial self-assembly of collagen peptide fibers, 12610-12611 © 2008 American Chemical Society such as cell adhesion and growth. This method makes use of the bonds formed between ligands and metal ions. Upon addition of metal ions to functionalized CMP strands, the complexes formed stable triple helices as well as a wide array of supramolecular structures.

In a (POG)9 CMP, the central hydroxyproline residue was replaced with a bipyridyl-modified lysine (KByp) and the chains self-assembled into triple-helices

(Figure 22A, B).112 Upon addition of Fe(II) metal ions, triple-helices joined together and 43 grew in the radial direction (Figure 22C).112 The Byp-modified CMP formed fibers, 3-5

μm in length, with extensive branching, as well as bundles of thin fibers with a 10 nm width (Figure 21D).112

Figure 23. A) Chemical structures of HisCol and IdaCol CMPs from Ref. 61. B) The peptide strands self-assemble into triple helices and adding metals binds the two CMPs. C-E) SEM images of the assemblies formed from C) Ni(II), scale bar = 1 μm D) Zn(II), scale bar = 500 nm E) Cu(II), scale bar = 500 nm. F) TEM images of the higher-ordered structures, scale = 700nm and insets at a higher magnification. G) TEM of the periodic banding suggests the length of the triple helices is 9 nm. Adapted with permission from the Journal of the American Chemical Society, 133/37, Pires, MM, Przybyla, DE, et al., Metal-mediated tandem coassembly of collagen peptides into banded microstructures, 14469-14471 © 2011 American Chemical Society

Higher-order structures were produced by a co-block CMP composed of a (POG)9 peptide flanked at both termini by metal-binding molecules, either histidine (HisCol) or iminodiacetic acid (IdaCol) (Figure 23A).61 These molecules formed higher-ordered structures when treated with Ni(II), Zn(II), and Cu(II) metal ions.61 The structures assembled from an alternating pattern of HisCol peptides followed by IdaCol peptides in a repeating sequence. The use of Ni(II) formed clusters of petal-like structures up to 500 nm long, whereas Zn(II) and Cu(II) formed less organized structures (Figure 23C-F).61

The alternating peptide-metal complexes promoted self-assembly of microstructures with

D-periodicity of 9-12 nm distance between banding gaps (Figure 23G).61

Metals such as Zn(II) and Cu(II) were also used to induce self-assembly of

(POG)n CMPs of various lengths (n = 5, 7, 9, 11) with an N-terminus nitrilotriacetic acid

(NTA) ligand and two histidine residues at the C-terminus (referred to as NTA CMP)

(Figure 24).113, 114 A length of n = 11 yielded 3-5 μm saddle-like structures and n = 9 yielded micrometer sized microflorettes (small petal-like structures). n = 7 formed a highly cross-linked 3-D mesh and n = 5 did not form a stable triple helix at room temperature.113, 114

Figure 24. A) Structure of the NTA CMP.114 B-E) SEM images of B) microflorettes, scale bar = 10 μm and C-E) saddle structures, scale bar = 3 μm. Adapted with permission from Langmuir, 28/4, Pires, MM, Lee, J, et al., Controlling the morphology of metal- promoted higher-ordered assemblies of collagen peptides with varied core lengths, 1993- 1997 © 2012 American Chemical Society

In another study, a 27-amino acid POG CMP containing three central bipyridine ligands self-associated into micrometer-sized, curved, disk-like structures.115

Fe(II)-promoted assembly yielded hollow spheres and Cu(II)-promoted assembly yielded fragile spheres that shattered easily.115 The length of the CMP sequence had an effect on the height of spherical disks.116 Longer peptides formed longer triple-helices, which in

45 turn, yielded taller disks. These disks were used as micro-cages to encapsulate and release fluorescently-labeled dextrans.116 Also, many polymer cages are made in harsh conditions and can damage or inactivate the biomolecule being delivered. This method used mild conditions to make the cages and therefore, there is a smaller chance of non- desired interactions.116

Three POG-based CMPs were designed with His2 at the N-terminus and NTA or

RGDS, a cell binding domain, on either the C-terminus or as the central residue.117 The first CMP, NTA-(POG)4-P(KByp)G-(POG)4-His-His (HBN), self-assembled into a 3-D mesh with addition of metal ions and was capable of cell encapsulation.118 The latter two,

NTA-(POG)4-PK(GGRGDS)G-(POG)4-His-His (HRGDSN) and GGRGDS-(POG)4-

P(KByp)G-(POG)4- His-His (HBRGDS), incorporated a cell-binding RGD sequence but in different locations on the chain. The RGD sequence of the latter two CMPs, was located either at the N-terminus or on the central residue. Adding Zn(II) to HBN yielded a fibrous scaffold with large pores, whereas adding Co(II), Cu(II), and Ni(II) yielded scaffolds with smaller pores ranging between 5 – 20 μm in diameter.117 Matrices of the

RGDS and the P(KByp)G peptides formed scaffolds and were able to gradually release growth factor.118 HBRGDS and HRGDSN were loaded with human epidermal growth factor (hEGF-His6) and seeded with human non-tumorigenic epithelieal cells (MCF10A cell line). These CMPs were able to induce adhesion and proliferation of the cells, thus showing the scaffolds were capable of releasing the hEGF-His6 that induced proliferation.118 MCF10A cells displayed spheroid, acinar structures in the matrix containing the HBRGDS CMP, with the RGDS sequence at the N-terminus.117 MCF10A

46 cells have a spheroid structure when the cells are healthy and are then capable of differentiating and grouping into acinar structures.117 An acinus is a cluster of cells that

Figure 25. A) Chemical structure of the catechol-containing CMP from Ref. 119. The CMP forms a triple helix due to the TRIS scaffold. B) Upon the addition of Fe3+ the stability of the triple helix was raised by 22 °C. Adapted with permission from the Journal of the American Chemical Society, 126/46, Cai, W, Kwok, SW, et al., Metal- assisted assembly and stabilization of collagen-like triple helices, 15030-15031 © 2004 American Chemical Society look like berries and are found in glands such as mammary glands.

Metal-triggered self-assembly was also accomplished for two hydroxyproline- lacking PPG peptides. This method was used to show that PPG peptides can be stabilized by metal-histidine coordination to form numerous microscale structures.62 One PPG CMP had histidine on the N- and C-termini, and another had histidine on both N- and C-termini as well as a centrally-located histidine.62 Upon addition of Zn(II), Cu(II), and other

47 metals, the CMPs exhibited nanofibers with a D-periodicity of ~9.5 nm, microscale spheres, and other supramolecular structures.62

Fe(III) is capable of forming an organic-inorganic complex with catechols, and this concept was used to add stability to the triple helix of a CMP but has yet to show any formation of higher-order structures.119 The CMP was designed with an N-terminal 2,3- dihydroxybenzoic acid that was attached to the CMP sequence with an ethylenediamine

119 linker (this is referred to as a TRIS scaffold) . The CMP sequence was (Gly-Nleu-Pro)6 and a catechol was placed at the C-terminus, which was linked to the CMP region also by ethylenediamine (Figure 25). Prior to adding the iron, the Tm of the TRIS-scaffold triple helix was 36 °C. Fe3+ formed a complex with the catechol groups and further stabilized

119 the triple helix of the CMP and raised the Tm to 58 °C.

4.9 Electrostatic Interactions

Collagen has many charged amino acids in its sequence which contribute to its supramolecular assembly. CMPs have been designed to aggregate by electrostatic interactions and form higher-ordered structures. These structures include hydrogels, fibers with a D-periodic spacing, and self-assembled 2-D nanosheets. A CMP was designed with a (PYG)4-(POG)4-(XOG)4 sequence where X was a C-terminal hydrophilic domain containing Glu, a negatively charged amino acid residue.63 The N-terminal was a

48 hydrophobic domain containing positively charged Arg in the Y position. Electrostatic interactions contributed to the formation fibers having a D-periodicity of ~18 nm and fibrils 3-4 μm long with 12-15 nm diameters (Figure 26).63 After an unspecified longer

Figure 26. A) Structure of the (PRG)4-(POG)4-(EOG)4 CMP. B) TEM image of fibers formed from the CMP with pointed tips. C) TEM displaying the 18 nm D-periodicity of the CMP fibers. D) TEM of the tactoidal end (circled) of a collagen fibril. A-C) Adapted with permission from the Journal of the American Chemical Society, 129/47, Rele, S, Song, Y, et al., D-periodic collagen-mimetic microfibers, 14780-14787 © 2007 American Chemical Society D) Adapted with permission from the Journal of Biological Chemistry, 254/22, Gelman, RA, Poppke, DC, and Piez, KA, Collagen fibril formation in vitro. The role of the nonhelical terminal regions, 11741-11745 © 1979 American Society for Biochemistry and Molecular Biology annealing time period, the CMP yielded fibrils with a micron-scale length and ~70 nm diameter with the same 18 nm periodicity.63 Also, the CMP fibers formed pointed tips

(Figure 26B) similar to collagen fiber tactoidal ends (meaning the tips of the fibers are non-helical).63, 120 (Figure 26D). Molecular dynamics simulations showed adjacent triple helices had a staggered orientation due to the electrostatic interactions between triple- helices. Simulations also showed that the linear oligomerization (the linear growth of triple-helices) was reinforced by hydrogen bonding between the N- and C-termini of linearly adjacent triple helices (meaning the interactions between the tail of one triple

49 helix and the head of another).63

63 Extending on the work above and using the (PYG)4-(POG)4-(XOG)4 sequence, two modified CMPs were designed, which formed 2-D nanosheets (Figure 27).121 In these CMPs, X was Glu, Y was (2S,4R)-4-aminoproline (Amp), and there were either

Figure 27. A) TEM of the NSI CMP showing the uneven distribution of the sizes of the nanosheets. Scale bar = 1 μm B) TEM of an individual nanosheet from NSI. Scale bar = 1 μm. C) TEM of an NSII nanosheet Scale bar = 500 nm. D) AFM image of an NSI nanosheet that has multiple layers. Scale bar = 200nm. E) AFM of an NSII single layer nanosheet. Scale bar = 200 nm. F) AFM height histogram of single-layered sheets of NSI (black) and NSII (white). Reprinted with permission from the Journal of the American Chemical Society, 136/11, Jiang, T, Xu, C, et al., Structurally defined nanoscale sheets from self-assembly of collagen-mimetic peptides, 4300-4308 © 2014 American Chemical Society four or seven central POG repeat units. Amp, like Hyp, has a Cϒ-exo ring puckering and thus, similar stereoelectronic effects.121 Also, the length of central POG repeat sequence mas modified as (POG)4 (referred to as NSI) or (POG)7 (referred to as NSII). The rate of assembly of the nanosheets depended on the concentration and molar mass of the peptides and ranged over the course of hours to weeks.121 NSI assembled in days at a concentration of 4 mg/mL and over the course of weeks when the concentration of

50 peptide was 2 mg/mL.121 On the other hand, NSII assembled into nanosheets over the course of hours with a concentration of 2.5 mg/mL.121 AFM was employed to observe the height of the nanosheets. Interestingly, 2 mg/mL concentrations of NSI formed thicker sheets than the higher concentration of 4 mg/mL of NSI as well as the 2.5 mg/mL solution of NSII.121 Neither NSI nor NSII formed a homogeneous population of nanosheets, meaning the films varied in size with a diagonal length ranging between 1-15

μm.121 The thickness of a single layer of NSI films was ~8.8 nm and the height of NSII films was ~12.3 nm.121 Gold nanoparticles bound with positively charged ammonium ions were used to test the presence of carboxylate groups from the C-terminus of the peptides were present on the surface of the NSII nanosheets. TEM and AFM verified that the gold nanoparticles bound to the surface.121 The researchers also biotinylated the N- terminus of NSII (denoted as NSII*), which also formed nanosheets and bound with streptavidin-tagged gold nanoparticles.121 The NSII* peptide was also found to bind on a biotinylated glass substrate to immobilize the sheets onto a surface.121

In addition to NSI and NSII, a third CMP (referred to as NSIII) was designed with

(2S, 4S)-4-aminoproline (amp), which has a Cϒ-endo rick puckering, thus mimicking the

122 stereoelectronic effects of Pro. The peptide sequence used was (amp-Hyp-Gly)4-(Pro-

Hyp-Gly)4-(Pro-Glu-Gly)4, where amp replaced Pro and Glu replaced Hyp. Unlike NSI and NSII, NSIII yielded a homogenous population of nanosheets, meaning the nanosheets of NSIII were all roughly the same size with a mean diagonal distance of ~679 nm and a height of ~9.6 nm.122 These measurements did not change between a peptide concentration range of 2-10 mg/mL.122

Two more CMPs were designed with sequences based on the CMP from Ref. 63, which contained Arg and Glu. Both of these CMPs contain the same sequence, (Pro-Arg-

123 + Gly)n1-(Pro-Hyp-Gly)4-(Glu-Hyp-Gly)n2. In the CMP labelled CP , n1 = 7 and n2 = 4.

In the CP- CMP, n1 = 4 and n2 = 7. CP+ and CP- both formed single layer nanosheets with positive and negative charges, respectively.123 Cationic and anionic gold nanoparticles were used to confirm the presence of surface charges.123 AFM was used to measure the dimensions of the nanosheets.123 CP- formed sheets with a height of ~9.4 nm and CP+ yielded sheets with a height of ~9.9 nm. The asymmetric distribution from the additional triads was incorporated to create extensions of the peptides that protrude from the surface of a single layer nanosheet. The negatively charged protrusions from a CP- nanosheet are able to form electrostatic interactions with CP+ protrusions in a ‘Lego-like’ assembly at the supramolecular level.123 This enabled the researchers to create a multilayer nanosheet, where a CP+ nanosheet was stacked between two nanosheets of CP- when the ratio of CP-/CP+ > 2.123 The height of the trilayer nanosheet was measured by

AFM to be ~34 nm.123

Further research expanded on the work of Chaikof 63 and a CMP was designed

124 with the sequence, (PKG)4(POG)4(DOG)4. This CMP had salt-bridged hydrogen bonds between Lys and Asp residues, instead of Arg and Glu. These bonds increased intermolecular interactions between both strands and triple-helices. This CMP was the first to self-assemble into a triple helix in a wide range of buffers and ionic strengths conditions.124 The CMP formed nanofibers, several hundred nanometers long with 4-5 nm width. The CMP also formed a hydrogel with good viscoelastic properties, meaning it

52 could maintain a molded shape. The hydrogel was also degraded by type IV collagenase at a similar rate to the degradation of a hydrogel prepared from a rat-tail.124 The hydrogel was shown to be noninflammatory, it promoted the adhesion and activation of platelets,

125 and was able to clot blood and plasma. The CMP was also found to have a 72 helical twist.126 CMPs using the same residues but with different domain arrangements showed that the formation of fibers occurred from a sticky-ended intermediate.126 This means that the charged pairs for the triple-helices and there are unsatisfied, glycine-based, backbone hydrogen bonds at the termini.126 These glycines are ‘sticky’ and enable triple helices to join together at the termini and ultimately yield fibers.126

Figure 28. A) TEM of a single detonation ND particle ~5 nm in diameter with inert sp3 diamond core. B) 5 nm diameter atomistic model of an ND displaying the many functional groups present on the surface that make selectivity in synthesis a challenge. Grey: sp3 diamond carbon. Green: sp3 amorphous carbon. Black: sp2 carbon. Red: oxygen. Blue: nitrogen. White: hydrogen. Adapted with permission from Biopolymers Peptide Science, 104/3, Knapinska, AM, Tokmina-Roszyk, D, et al., Solid‐phase synthesis, characterization, and cellular activities of collagen‐model nanodiamond‐ peptide conjugates, 186-195 © 2015 John Wiley and Sons

To study the liquid-crystalline behavior of CMPs, a triblock CMPs was designed

- with the sequence (Glu)5 (GXPGPP)6-(Glu)5, where (Glu)5 was used to cap the peptide to promote solubility and triple helix stability.127 When X = Pro or Ala, films that formed 53 from evaporation of these CMP solutions displayed banded spherulites with a cholesteric- like twist. When X = Ser, the films showed no organization.127

4.10 Nanodiamond-CMP Conjugates

Nanodiamonds (NDs) (Figure 28) have a low production cost and have applications in drug delivery such as sustained drug release from films that are embedded with NDs.128 Covalently attaching NDs to peptides is complicated because the reaction is not selective as to where the NDs will bind with a peptide.128 Fields and coworkers were able to selectively, covalently link NDs with the N-terminus of a POG CMP that mimicked α1(I)fTHP, an integrin-binding sequence found in type I collagen.128 This ND-

CMP conjugate formed stable triple helices but has not yet been shown to form higher- ordered structures. The ND-CMPs were shown to have biological relevance; Chinese hamster ovary cells (CHO-K1) and BJ fibroblasts were able to adhere to and were viable on the ND-CMPs.128

4.11 Polymerized-CMPs and CMP-Dendrimers

This final section reviews polymerized-CMPs and CMP-dendrimers. These conjugated CMPs generally form higher-ordered structures such as fibers hydrogels due

Figure 29. CMP-polymer designed by Hartgerink and coworkers using native chemical ligation.131 A) Schematic of the mechanism of native chemical ligation showing the intermediate thioester formed from the N-terminal cysteine. B) The N-terminal cysteine and C-terminal thioester polymerize through native chemical ligation. Two reactive groups are shown in the squares. C) To control the polymerization, hydrolysis of the thioester group prevents the polymer chain from growing uncontrollably. Reprinted with permission from Macrmolecules, 38/18, Paramanov, SE, Gauba, V, and Hartgerink, JD, Synthesis of collagen-like peptide polymers by native chemical ligation, 7555-7561 © 2005 American Chemical Society to the high molecular weight and inherent nature of polymers and dendrimers. This section only contains a few examples of polymerized-CMPs and for detailed reviews of polymerized-CMPs and polymer-CMP conjugates see Luo and Kiick47 and He and 55

Theato.129

As mentioned in Section 4.2, high molecular weight (HMW) POG CMP polypeptides were yielded from a polycondensation polymerization of (POG)10 peptides.65 These HMW CMPs formed triple-helical and aggregated nanofiber structures with a 10 nm diameter.65 Triple helices and fibers of POG and POG-based CMPs have also been created by the use of native chemical ligation, in which a C-terminal thioester was polymerized with an N-terminal cysteine (Figure 29).130, 131 This yielded a HMW polymeric-CMPs on the order of hundreds of kilodaltons (kDa), that formed a dense network of fibers that were microns in length with a 10-20 nm diameter.131

A reversible addition-fragmentation chain transfer polymerization (RAFT) was employed to design a triblock copolymer. A CMP with the sequence (GPP)3-

GPRGEKGERGPR-(GPP)3-GPCCG was polymerized with poly-diethylene glycol methyl ether methacrylate at both the N- and C-termini.132 The same CMP sequence was used as a collagen-based gene transfer delivery system for applications in gene therapy.

The CMP acted as a tether to bind a DNA-polyplex with collagen-based 2-D films and 3-

D gels.133 The CMP was able to act as a tether for two weeks on 2-D films and for a month on 3-D gels.133 This time frame was longer than previously used methods of tethering, which only lasted a few days.133

A poly(ethylene glycol)-CMP macromonomer was developed, where the CMP

134 sequence was NH2-(POG)7-Tyr-OH. Photopolymerizing the APEG CMP with poly(ethylene oxide) diacrylate (PEODA) yielded a CMP/PEODA hydrogel.

Chondrocytes were encapsulated in the hydrogel to test its use as a tissue engineering

56 scaffold (Figure 30).134 Production of glycosaminoglycans, a polysaccharide unit of proteoglycans that is a major component of cartilage extracellular matrices, was measured. It was found that the CMP/PEODA hydrogel enhanced the tissue production of chondrocytes.134

Figure 30. A, B) In a PEODA hydrogel, chondrocytes secrete collagen, however in the CMP/PEODA hydrogel, interactions between the CMP and collagen enabled the hydrogel to retain cell-secreted collagen and promoted a fast accumulation of extracellular matrix products.134 C) Fluorescence micrograph of chondrocytes embedded in the CMP/PEODA hydrogel. Adapted with permission from Biomaterials, 27/30, Lee, HJ, Lee, JS, et al., Collagen mimetic peptide-conjugated photopolymerizable PEG hydrogel, 5268-5276, © 2006 Elsevier

A CGG-(POG)8 CMP sequence was linked to an 8-arm star poly(ethylene glycol) polymer (PSP) to form hydrogels (Figure 31).135 An 8% concentration of PSP-CMP hydrogel exhibited a higher storage modulus than a 4% concentration of PSP-CMP hydrogel. The PSP-CMP hydrogels had honeycomb-like structures with pore sizes ranging from 5-25 μm.135 After thermal annealing, the 8% concentration had significantly smaller pore sizes of approximately 2 μm.135 The pore sizes of the 4% concentration hydrogel remained basically unchanged after thermal annealing.135 The PSP-CMP hydrogels were capable of encapsulating human mesenchymal stem cells (hMSCs)

(Figure 31D, E) and were non-toxic to the cells.135

The CGG-(POG)n sequence was further probed, where n = 7, 8, or 9 and were conjugated to a PEG 8-arm star.136 All three of these PSP-CMPs formed hydrogels with porous networks at room temperature. Pore sizes averaged between 4-10 μm but within a polydisperse range of 2-25 μm. Increasing repeat units of (POG)n resulted in a higher Tm

Figure 31. A) Schematic showing the addition of CMP to an 8-arm star poly(ethylene glycol) to form a hydrogel for the encapsulation of human mesenchymal stem cells. The insert is an SEM image that shows the porous structure of the hydrogel. B, C) SEM of the hydrogel at two different magnifications. D) Fluorescence image of the human mesenchymal stem cells encapsulated in the hydrogel. E) Cryo-SEM image of the hMSCs in the hydrogel. Reprinted with permission from Macromolecular Bioscience, 11/10, Rubert Perez, CM, Panitch, A, and Chmielewski, J, A collagen peptide‐based physical hydrogel for cell encapsulation, 1426-1431 © 2011 John Wiley and Sons in both the triple helix of the CMP itself as well as in the PSP-CMP hydrogels. The stiffness of the hydrogels was not affected by the number of POG repeats.136

Two CMPs were designed with either a Gly-Pro-Nleu or a Gly-Nleu-Pro CMP sequence and were conjugated with tris-(carboxyethoxymethyl)-aminomethane, which is another TRIS scaffold (referred to from here as TRISA).137 Three TRISA-CMP molecules joined together around a trimesoyl chloride core and formed a dendritic

58 structure with stable triple helices (Figure 32).137 In accordance with earlier work,35, 37 the triple-helices of Gly-Pro-Nleu TRISA-CMP were less stable than those of the Gly-Nleu-

Pro TRISA-CMP.137

To increase the Tm of a Gly-Pro-Nleu dendrimer, a polyamidoamine (PAMAM)

dendrimer was conjugated with a Gly-Pro-Nleu repeat sequence.138 The Gly-Pro-Nleu-

PAMAM dendrimer yielded triple helices with a Tm of 25 °C, slightly more stable than

138 the Gly-Pro-Nleu-TRIS dendrimer with Tm = 22 °C. Extending on this work, a CMP-

PAMAM dendrimer was designed with POG repeat units, the GFOGER sequence, and

Figure 32. Schematic showing the structure of the TRISA-CMP and the three triple helices bound around a trimesic acid core.137 Reprinted with permission from the Journal of the American Chemical Society, 124/51, Kinberger, GA, Cai, W, and Goodman, M, Collagen mimetic dendrimers, 15162-15163 © 2002 American Chemical Society either APQQAE (CMD-Q) or EDGFFKI (CDM-K) sequences at the C-terminus (Figure

33).139 The latter are sequences that interact with tissue transglutaminase (tTGase), an enzyme that mediates cross-linking. These CMP dendrimers were found to be non- cytotoxic and promoted the adhesion of L929 fibroblast cells and human hepatocarcinoma cells (Hep3B).139

Hydroxyproline-free CMPs, (PPG)5 and (PPG)10, were also linked with PAMAM 59

140-142 dendrimers and also yielded stable triple-helices. The (PPG)10-PAMAM dendrimer

140-142 had higher stability than the (PPG)5-PAMAM dendrimer. The (PPG)5- and (PPG)10-

PAMAM dendrimers also formed temperature-dependent hydrogels without the use of

Figure 33. A) Schematic showing the preparation of PAMAM dendrimer-bound CMPs containing the GFOGER sequence.139 B-D) Images of the actin cytoskeletal structure of Hep3B cells on B) calf-skin collagen, C) enzymatically crosslinked CMD-Q and CDM-K CMPs and on D) CDM-K. Adapted with permission from Biomaterials, 29/20, Khew, ST, Yang, QJ, and Tong, YW, Enzymatically crosslinked collagen-mimetic dendrimers that promote integrin-targeted cell adhesion, 3034-3045 © 2008 Elsevier cross-linking and were capable of thermosensitive drug release of rose bengal, a medicine used in a variety of applications.140 This concept was then applied to POG CMPs. A

(POG)10 CMP was conjugated with a PAMAM dendrimer a heat-induced hydrogel at 40

143, 144 °C. By comparing the stability of (POG)10-PAMAM to (POG)n-PAMAM, where n

= 2, 5, and 8, they found triple helix stability increased with the number of POG

144 repeats. (POG)5-PAMAM and (POG)10-PAMAM exhibited temperature dependent

144 drug release of rose bengal, similarly to the (PPG)n-PAMAMs. The (POG)10-PAMAM dendrimer formed a hydrogel at 45 °C, compared to the (PPG)10-PAMAM which formed a hydrogel at 40 °C. This is because the POG sequence provides more stability than the

PPG sequence.

CHAPTER 5

CONCLUSION

Collagen mimetic peptides have been used to study the physical, chemical, and biological properties of collagen. CMPs have been used to understand how the amino acid sequence enables the formation of triple-helices of collagen and its self-assembly into higher-ordered fibers and fibrils. CMPs can be tailored to have specific sequences to target and promote specific biological functions. CMPs have also been designed to form many supramolecular structures with a range of applications.

CMPs have a general design principle based on POG because they confer the most stability to triple-helices and can self-assemble into nanofibers. PPG sequences are also studied as the major sequence because recombinant peptides can be produced by E. coli but they do not provide as much stability as the POG sequence nor do they form nanofibers on their own. Cysteine knots and electrostatics interactions are employed to further add stability to POG and PPG triple-helices and contribute to the formation of nanofibers and hydrogels among other higher-ordered structures. Other methods to stabilize triple-helices and form higher order structures include metal-induced self-

assembly, polymerization of CMPs, conjugation with polymers and dendrimers, and aromatic interactions.

CMPs have been shown to have applications but the field is still in its infancy.

Many CMPs have been shown to promote cell adhesion and viability but there are very few CMPs that have been shown to induce cellular responses and mechanisms. For example, MC3T3-E1 preosteoblast have been found to be viable on specific CMPs.

However, no work has shown if the cells can differentiate on these surfaces or if CMPs can induce differentiation. Cell adhesion studies are important and necessary but to be used as robust biofunctional materials there needs to be significantly more research in the field.

One of the major claims and goals of CMPs is to replace collagen as a biomaterial. Many authors make the argument that collagen from bovine or other sources can induce an inflammatory response in patients and therefore there is a need for synthetic alternatives. This claim is not justifiable as porcine and bovine collagens are often and widely used in reconstructive surgery, cosmetic surgery, among many other medical procedures with low risk. The only way CMPs could realistically replace collagen is if they are shown to be better than collagen in some way and that has yet to happen. It is the opinion of the author that CMPs may best be suited for understanding the structures and stability of collagen and triple-helices but have little practical application.

CHAPTER 6

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