Beta-sheet peptide-mediated self-assembly of HPMA Copolymers into nanostructured biomaterials

The use of ?-sheets as building blocks for biomaterials is already firmly established. In particular, self-assembled ?-sheet peptides are promising for engineering new fibrous nanostructures and hydrogels. Peptide-synthetic polymer hybrids are especially attractive since they combine the advantages of biomolecular recognition and functional properties of peptides with the low cost and easy fabrication of polymers. Significant developments in the area have included ?-sheet fibrillar networks, and selfassembled hybrid hydrogels, which add further control and utility to these systems. The studies described in this dissertation dealt with the design and evaluation of novel nanofibrous and hydrogel materials based on poly(HPMA)-?-sheet copolymers and their application as scaffolds for bone tissue engineering. In the first part of this research, the effect of conjugating poly(HPMA) to a ?-sheet peptide via thiol-maleimide chemistry was estimated. The ability of the peptide to adopt a ?-sheet conformation could be imposed in the hybrid at basic pH, through electrostatic interactions between the oppositely charged amino acid residues in the sequence. Hierarchically organized structures, such as micrometer long fibrils, were obtained. In the second part, formation of fibril-like nanostructures was demonstrated for ?-sheet peptides conjugated as grafts to poly(HPMA).

BETA-SHEET PEPTIDE-MEDIATED SELF-ASSEMBLY OF HPMA COPOLYMERS INTO NANOSTRUCTURED BIOMATERIALS by Larisa Cristina Wu A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Bioengineering The University of Utah December 2010 Copyright © Larisa Cristina Wu 2010 All rights reserved The University of Utah Graduate School STATEMENT OF DISSERTATION APPROVAL The dissertation of Larisa Cristina Wu has been approved by the following supervisory committee members: Jindřich Kopeček , Chair 08/12/2010 Date Approved Vladimir Hlady , Member 08/12/2010 Date Approved Hamid Ghandehari , Member 08/12/2010 Date Approved Russell J. Stewart , Member 08/12/2010 Date Approved Jiyuan Yang , Member 08/12/2010 Date Approved and by Richard D. Rabbitt , Chair of the Department of Bioengineering and by Charles A. Wight, Dean of The Graduate School. iv ABSTRACT The use of β-sheets as building blocks for biomaterials is already firmly established. In particular, self-assembled β-sheet peptides are promising for engineering new fibrous nanostructures and hydrogels. Peptide-synthetic polymer hybrids are especially attractive since they combine the advantages of biomolecular recognition and functional properties of peptides with the low cost and easy fabrication of polymers. Significant developments in the area have included β-sheet fibrillar networks, and self-assembled hybrid hydrogels, which add further control and utility to these systems. The studies described in this dissertation dealt with the design and evaluation of novel nanofibrous and hydrogel materials based on poly(HPMA)-β-sheet copolymers and their application as scaffolds for bone tissue engineering. In the first part of this research, the effect of conjugating poly(HPMA) to a β-sheet peptide via thiol-maleimide chemistry was estimated. The ability of the peptide to adopt a β-sheet conformation could be imposed in the hybrid at basic pH, through electrostatic interactions between the oppositely charged amino acid residues in the sequence. Hierarchically organized structures, such as micrometer long fibrils, were obtained. In the second part, formation of fibril-like nanostructures was demonstrated for β-sheet peptides conjugated as grafts to poly(HPMA). The polymer had a shielding effect, decreasing the peptide grafts sensitivity to temperature and pH variations. The tendency of β-sheets to form hydrogels was preserved in the copolymer depending on the concentration, graft density, and iv incubation time. Finally, the last part of this research attempted to explore the ability of a hybrid hydrogel self-assembled from copolymers of poly(HPMA) and complementary β- sheet grafts to act as scaffolds for bone tissue engineering. The hydrogel displayed anisotropic porosity, thus, it provided surfaces characterized by epitaxy that favored template-driven mineralization of hydroxyapatite, and support for preosteoblast cells. Although attachment did not occur, long-term viability of cells and proliferation indicated that the hybrid hydrogel is not cytotoxic, therefore, once optimized, could be used as a bone scaffold. In summary, we have presented novel β-sheet-based hybrid nanostructures and hydrogels that could be applied successfully in regenerative medicine. Such an approach may lead to the development of new materials for drug delivery, wound healing or other tissue engineering applications. This dissertation is dedicated to my daughter Lorena Ailan, the guiding light in my life i TABLE OF CONTENTS ABSTRACT ....................................................................................................................... iii LIST OF TABLES ...............................................................................................................x LIST OF FIGURES ........................................................................................................... xi ACKNOWLEDGMENTS ............................................................................................... xiv Chapter 1. INTRODUCTION ..........................................................................................................1 1.1. Self-assembled peptides as platform for the formation of nanostructures and hydrogels ....................................................................................................1 1.2. The β-sheet motif .............................................................................................3 1.3. Nanofibrous materials based on hybrid copolymer systems .............................7 1.3.1. Selection of polymer ..........................................................................9 1.3.1.1. Poly(ethylene glycol) .........................................................10 1.3.1.2. Poly[N-(2-hydroxypropyl)methacrylamide] ......................11 1.3.2. Nanofibrous hybrid block copolymers .............................................11 1.3.3. Nanofibrous hybrid graft copolymers ..............................................13 1.3.4. Hybrid hydrogels .............................................................................13 1.4. Biomineralization in β-sheet self-assembled materials ...................................16 1.4.1. Hydroxyapatite .................................................................................18 1.4.2. Bone mineralization .........................................................................18 1.4.3. Biomineralization in β-sheet nanofibers ..........................................21 1.4.4. Biomineralization in hydrogels ........................................................24 1.5. Hybrid hydrogels for bone tissue engineering ................................................26 1.6. Statement of objectives ...................................................................................30 1.7. References .......................................................................................................32 2. SELF-ASSEMBLING DIBLOCK COPOLYMERS OF POLY(HPMA) AND A β-SHEET PEPTIDE .................................................................................................45 2.1. Summary .........................................................................................................45 2.2. Introduction .....................................................................................................46 2.3. Experimental part ............................................................................................49 vii 2.3.1. Materials ..........................................................................................49 2.3.2. Peptide synthesis and purification ...................................................50 2.3.3. Synthesis and purification of β-sheet peptide-poly(HPMA) diblock copolymers ..........................................................................51 2.3.4. Circular dichroism spectroscopy ......................................................54 2.3.5. Congo Red binding studies ..............................................................55 2.3.6. Transmission electron microscopy ..................................................56 2.3.7. Atomic force microscopy .................................................................56 2.4. Results and discussion ....................................................................................57 2.4.1. Synthesis of P11 peptide and P11-poly(HPMA) conjugates ...........57 2.4.2. Circular dichroism spectra of P11 peptide and P11-poly(HPMA) conjugates...........................................................58 2.4.3. Congo Red binding study .................................................................63 2.4.4. Morphology of the P11 peptide and P11-poly(HPMA) conjugates self-assemblies ...............................................................64 2.5. Conclusion ......................................................................................................70 2.6. Acknowledgements .........................................................................................71 2.7. References .......................................................................................................71 3. SELF-ASSEMBLED HYDROGELS FROM POLY(HPMA) GRAFTED WITH β-SHEET PEPTIDES ........................................................................................75 3.1. Summary .........................................................................................................75 3.2. Introduction .....................................................................................................76 3.3. Experimental part ............................................................................................79 3.3.1. Materials ..........................................................................................79 3.3.2. β-Sheet peptide synthesis .................................................................80 3.3.3. Synthesis and purification of poly(HPMA) grafted with β-sheet peptide [poly(HPMA)-g-CGGBeta11] ................................81 3.3.4. Circular dichroism spectroscopy ......................................................83 3.3.5. Fourier transform infrared spectroscopy ..........................................83 3.3.6. Thioflavin T binding studies ............................................................84 3.3.7. Transmission electron microscopy ..................................................84 3.3.8. Hydrogel preparation .......................................................................85 3.3.9. Scanning electron microscopy .........................................................85 3.3.10. Microrheology................................................................................86 3.4. Results and discussion ....................................................................................86 3.4.1. β-Sheet peptide design .....................................................................86 3.4.2. Synthesis and characterization of poly(HPMA) grafted with β-sheet peptide .................................................................................88 3.4.3. Characterization of hydrogels self-assembled by β-sheet peptide domains ...............................................................................97 3.5. Conclusions ...................................................................................................104 3.6. Acknowledgements .......................................................................................105 3.7. References .....................................................................................................105 viii 4. HYBRID HYDROGELS SELF-ASSEMBLED FROM GRAFT COPOLYMERS CONTAINING COMPLEMENTARY β-SHEETS AS SCAFFOLDS FOR BONE TISSUE ENGINEERING ..............................................................................109 4.1. Summary .......................................................................................................109 4.2. Introduction ...................................................................................................110 4.3. Experimental part ..........................................................................................114 4.3.1. Materials ........................................................................................114 4.3.2. Complementary β-sheet peptides synthesis ...................................115 4.3.3. Synthesis of complementary poly(HPMA) copolymers grafted with β-sheet and RGD peptides .....................................................116 4.3.4. Circular dichroism spectroscopy ....................................................118 4.3.5. Congo Red binding studies ............................................................118 4.3.6. Thioflavin T binding studies ..........................................................120 4.3.7. Transmission electron microscopy ................................................120 4.3.8. Hydrogel preparation .....................................................................121 4.3.9. Rheological characterization ..........................................................121 4.3.10. Fourier transform infrared spectroscopy ......................................122 4.3.11. Scanning electron microscopy .....................................................122 4.3.12. MC3T3-E1 cells culture ...............................................................122 4.3.13. Cell viability assay .......................................................................123 4.4. Results and discussion ..................................................................................124 4.4.1. Design of hybrid hydrogels based on complementary poly(HPMA) copolymers grafted with β-sheets and RGD ............124 4.4.2. Characterization of fibers self-assembled from complementary poly(HPMA) copolymers grafted with β-sheets and RGD .....................................................128 4.4.3. Characterization of hydrogels self-assembled from complementary poly(HPMA) copolymers grafted with β-sheets and RGD .....................................................130 4.4.4. Mineralization in hybrid hydrogels ................................................135 4.4.5. Hybrid hydrogels as scaffolds for bone cells .................................142 4.5. Conclusions ...................................................................................................145 4.6. Acknowledgements .......................................................................................146 4.7. References .....................................................................................................146 5. CONCLUSIONS AND FUTURE WORK .................................................................151 5.1. Specific Aim 1. .............................................................................................152 5.2. Specific Aim 2 ..............................................................................................153 5.3. Specific Aim 3. .............................................................................................155 5.4. Future work ...................................................................................................157 5.4.1. Optimization of poly(HPMA)-g-β-sheet,RDG design to improve the preosteoblast cells attachment to the hybrid hydrogel scaffold .....................................................................................157 5.4.2. Determination of bioactivity in hybrid hydrogels scaffolds containing β-sheet and RGD peptides ......................................158 ix 5.4.3. In vivo evaluation of hybrid hydrogel scaffold ..............................159 5.4.4. References ......................................................................................159 xii LIST OF TABLES Table 1.1. Biological apatite minerals .........................................................................................19 3.1. Peptide structures. .......................................................................................................87 4.1. Peptide content in complementary poly(HPMA)-g-Beta11,RGD copolymers ........119 4.2. Complementary peptide structures ...........................................................................125 xiii LIST OF FIGURES Figure 1.1. The β-sheet motif; diagram of parallel and antiparallel arrangements ........................4 1.2. Hierarchical self-assembly of β-sheets-forming peptides .............................................6 1.3. Conjugation approaches to synthesize β-sheet peptide-polymer conjugates ................8 1.4. Alignment of the HA crystals with collagen/β-sheet fibers ........................................17 1.5. Mechanism of HA nucleation on the surface of self-assembled β-sheets incubated in SBF ........................................................................................................................23 2.1. The hypothesized self-assembly of β-sheet-poly(HPMA) diblock copolymer ...........48 2.2. Synthesis of β-sheet P11 peptide-poly(HPMA) diblock copolymers via thiol-maleimide coupling reaction ......................................................................................59 2.3. CD spectra of P11, P11-poly(HPMA)2k and P11-poly(HPMA)5k ............................61 2.4. Congo Red binding assay and differential spectra ......................................................65 2.5. TEM images of negatively stained P11, P11-poly(HPMA)2k and P11- poly(HPMA)5k ...........................................................................................................66 2.6. AFM images of P11, P11-poly(HPMA)2k and P11-poly(HPMA)5k. .......................69 3.1. Proposed model of poly(HPMA)-g-β-sheet hybrid hydrogel formation. ...................78 3.2. Synthesis of poly(HPMA)-g-CGGBeta11 graft copolymer and its self-assembly into a hydrogel via association of pendant β-sheet peptide strands ...........................89 3.3. Temperature-dependent and pH-dependent CD spectra of Beta11 and poly(HPMA)-g-CGGBeta11 graft copolymer ............................................................91 3.4. FTIR spectra of Beta11 and poly(HPMA)-g-CGGBeta11 graft copolymer ...............93 3.5. ThT fluorecence emission spectra and differential spectra ........................................96 xii 3.6. TEM and SEM of Beta11 and poly(HPMA)-g-CGGBeta11 fibrils and gels .............98 3.7. Mean square displacement plots as a function of lag time for PS particles in Beta11 and poly(HPMA)-g-CGGBeta11 solutions/gels ...........................................101 3.8. Frequency-dependent linear viscoelastic moduli for PS particles in Beta11 and poly(HPMA)-g-CGGBeta11 solutions/gels .......................................................103 4.1. Proposed model of hybrid hydrogel formation from self-assembled poly(HPMA)-g-β-sheet complementary copolymers ...............................................113 4.2. Synthesis of β-sheet and RGD conjugated poly(HPMA) copolymers ......................127 4.3. CD spectra of Beta11A:Beta11B and poly(HPMA)-g-Beta11A,RGD: poly(HPMA)-g-Beta11B,RGD in water at pH 7 ......................................................129 4.4. CR binding assay and differential spectra and ThT binding assay fluorescence emission spectra .......................................................................................................131 4.5. TEM images of Beta11A:Beta11B and poly(HPMA)-g-Beta11A,RGD: poly(HPMA)-g-Beta11B,RGD fibrils.......................................................................132 4.6. Dynamic frequency sweeps showing the dependence of G' and G" from 0.1 to 10 rad·s-1 of co-assembled peptides and co-asssembled copolymers hydrogels at different concentrations ............................................................................................134 4.7. Time sweeps showing the evolution of G' and G" as a function of time for co-assembled peptides and co-asssembled copolymers hydrogels at 3 wt.% concentration ................................................................................................136 4.8. SEM micrographs of Beta11A:Beta11B and poly(HPMA)-g-Beta11A,RGD: poly(HPMA)-g-Beta11B,RGD hydrogels before and after mineralization and EDS spectra of minerals grown on hybrid hydrogels ........................................139 4.9. ATR-FTIR spectra of gels mineralized in SBF ........................................................141 4.10. Confocal images of preosteoblasts seeded and embedded in Beta11A:Beta11B and poly(HPMA)-g-Beta11A,RGD:poly(HPMA)-g-Beta11B,RGD hydrogels at 1 week-culture ....................................................................................143 ACKNOWLEDGEMENTS I would like to thank my advisor, Dr. Jindřich Kopeček, for guidance and continuous support throughout my Ph.D. studies. Your enthusiasm for research, determination, and hard work were inspirational and have touched my personal and professional development. Without your direction, this dissertation would not have been possible. To Pavla, thank you for your help and consideration. Your kindness made my time in the lab even more enjoyable. Jane, thank you for everything you have done for me. I am very appreciative of the help and friendship you offer me every single day. I also thank my other committee members, Dr. Vladimir Hlady, Dr. Hamid Ghandehari, and Dr. Russell J. Stewart for being available, providing insight, technical expertise and support during this project. Special thanks to Dr. Chun Wang for serving as an external reviewer of my dissertation. This research was funded by the National Institutes of Health under grants EB005288 and GM69847. To my parents and my brother, thank you for loving, and supporting me through good and bad times during my pursuit of higher education over the seas. This dissertation is your accomplishment as well. To my husband, Kuangshi, thank you for being my companion on this exciting Ph.D. journey and in life. I look forward to our next adventure. To my friends and colleagues, thank you for being there. I am grateful for all I received during these last years. All these people shaped me as the person I am right now. It has been the best time of my life. 1 CHAPTER 1 INTRODUCTION 1.1. Self-assembled peptides as platform for the formation Molecular self-assembly, by definition, is the spontaneous organization of chemical and/or structural complementary molecules into well-defined arrangements formed and maintained by noncovalent interactions [1]. With the increased demand for structural and functional control at the molecular level, the self-assembling peptides and proteins, ubiquitous in nature, emerged as the preferred building blocks for creating new materials from the bottom up. The term "peptide Lego®" has been used to describe the spontaneous self-assembly of peptides into well-formed nanostructures at the molecular level [2, 3]. Self-assembly of peptides is highly specific, stimuli-responsive, and in the same time, it provides a precise control of the material structure and properties [4]. It has been observed that the highest degree of molecular recognition occurs in natural folding motifs such as α-helices, coiled-coils, β-sheets and turns. Structures forming coiled-coils and β-sheets have been employed to mediate the self-assembly of peptides, as well as peptide-polymer hybrid systems. The incorporation of these motifs into hybrid system leads to materials with superior properties when compared to individual components. For example, the peptide segment enhances the control over the nanoscale structure of the of nanostructures and hydrogels 2 synthetic segment, whereas the synthetic segment improves biocompatibility, and prevents degradation and loss of peptide function [5]. In particular, the β-sheet motif might be one of the most suitable organization motifs for peptide-guided assembly of synthetic polymers. The β-sheet-forming peptide domains have an extraordinary ability to impose their self-assembly properties on the overall hybrid system [6-9]. This process is responsive to changes in pH, temperature, solvents and is driven by intermolecular and intramolecular interactions which enable hierarchical, supramolecular organization in a concentration dependent manner [10]. Non-covalent interactions established in β-sheets, including electrostatic, hydrophobic, π- stacking, hydrogen bonding, and steric contributions, although weak and negligible when isolated, promote self-assembly into well-defined, stable macroscopic structures [11, 12]. Typically, they give rise to 2-D fiber nanostructures, which can further entangle into 3-D networks. As a result, β-sheets have been used not only to mediate the self-assembly of hybrid block [6-9, 13, 14] and graft copolymers [15, 16] into nanofibrous materials, but also into hydrogels [17-19]. Interestingly, the organization of the hybrid copolymers is very similar to that of the native peptides, which indicates that the self-assembly is driven mainly by the biorecognition established between the peptide architectural motifs. Conjugation with a synthetic polymer segment does not disrupt the natural tendency of the β-sheets-forming peptides to fold. Moreover, it can prevent lateral aggregation of the fibers and lead to soluble materials [6, 9], particularly important in case of the polymer-coated β-amyloid fibers that exhibit a strong reduction of amyloid plaques, associated with, e.g., Alzheimer's and Parkinson's diseases. 3 The β-sheet motif was identified by Pauling and Corey in the early 1950' [20]. β- Sheets consist of multiple extended polypeptide chains, referred to as β-strands, connected by a network of hydrogen bonds between the backbone amides and carbonyls. β-Sheets can be oriented so all their C-termini are at one end of the structure, described as a parallel structure, or so that N- and C-termini alternate, in an antiparallel arrangement, as shown in Figure 1.1. 1.2. The β-sheet motif β-Sheets are well known for their ability to self-assemble into fibers characterizing amyloid diseases like Alzheimer's and Parkinson's [21] and structure of silk protein [22]. The basic architecture present in most β-sheets consists of alternating hydrophobic and hydrophilic amino acid residues [23]; as a consequence of this pattern, they display, when self-assembled, a hydrophobic face and a hydrophilic one [11, 24]. Fibrous systems, therefore, result from the aggregation of sheets in the attempt to bury the hydrophobic faces from the contact with the surrounding water. Although well-defined rules have not been established so far, the design of synthetic β-sheet peptides, so called "amyloid-like" peptides, mainly exploits this intricate interplay between self-assembly and solubility in water. In fact, the characteristic poor solubility of the β-sheets in water is the consequence of the intermolecular peptide-peptide networking through interactions established between hydrophobic faces [25]. Other contributions come from cross-strand attractions between charged amino acids, lateral recognition between adjacent β-strands, and avoidance of edge-to-edge coulombic attractions between β- sheets by chemically blocking the N- and C-termini [26-28]. 4 N N N O O O N N N R R R H H H R N R O H R R R R N R O O O O H H H H antiparallel N N N O O O R R R H H H R N R O H N N N O O O R R R H H H R N R O H parallel Fig. 1.1: The β-sheet motif; diagram of parallel and antiparallel arrangements 5 Different hierarchical structures can be formed from β-sheet peptides in solution, with increasing concentration. The peptides self-assemble successively (upon reaching critical concentration values) into tapes, ribbons, fibrils and fibers as illustrated in Figure 1.2 [29]. The fibrillization occurs, after a lag phase, in an autocatalytic process characterized by nucleation and growth steps [30, 31]. The lag phase can be easily eliminated by seeding, that is, by addition of the preformed aggregated β-sheet structures, followed by incubation [32]. The fibrillization process debuts with the self-assembly of two complementary β-sheets into a ribbon or a "steric zipper" at high enough concentration giving rise to the spine of a fibril [10, 33, 34]. Further aggregation leads to fibers that can vary in size ranging from few nanometers to several micrometers in length. Investigation of the structure by X-ray diffraction and solid state NMR revealed much needed information about these β-sheet hierarchical assemblies. According to experimental studies, all amyloid-like peptides share a common structural feature: in a fibril/fiber, the β-strands are arranged perpendicular to the main axis, in the so-called "cross-β" structure [21, 30]. Moreover, the repeat distance along the polypeptide chain is 6.9 Å, the distance between two β-strands assembled in a sheet is 4.8 Å, whereas the inter-sheet distance or the stacking periodicity of the β-sheets is 10-11 Å [35]. The high level of order within the amyloid-like fibrils/fibers confers stability; therefore the structures are typically preserved at extreme conditions of pH, temperature and pressure, in both aqueous and organic solvents. They also have remarkable mechanical properties and precise spacing of chemical functionalities which make them suitable for applications such as high strength components in composite materials, nanowires, fibers for medical applications as scaffolds, and components for biosensors or actuators [31, 36]. 6 = β-strand β-sheet tape ribbon fibril fiber Figure 1.2: Hierarchical self-assembly of β-sheets-forming peptides 7 As a result of the applications the nanofibrous materials have in fields such as nanotechnology, materials science and medicine, preparation of the polymer nanofibers via peptide-guided self-assembly attracted lately the interest of research activity of numerous investigators. The focus has been on the structure of the polymer that can be programmed at the nano level by controlling the interactions between the peptide segments. The key challenge in this area is to modify the β-sheet peptide fibril with the polymer segment so that the structural properties and fibril characteristics are retained. As of now, two main approaches of the modification, or conjugation, can be identified (Figure 1.3): 1.3. Nanofibrous materials based on hybrid copolymer systems - the "grafting from" strategy that employs polymerization techniques by which the polymer segment is synthesized in the presence of the β-sheet peptide, and - the "grafting to" strategy which is based on coupling techniques involving the reaction between the preformed synthetic polymer and specific functional groups on the β-sheet peptide [36-38]. Site-selective conjugation is preferred over random conjugation because it allows precise control over the protein/polymer ratio and does not have a negative influence on the properties of the polymer or the peptide. As a result, the structure of the peptide-polymer conjugates can be controlled to match the application for which they are intended. The most common architecture is the head-to-tail conjugate, or linear block copolymer, having one polymer and one peptide block. Alternatively, a comb-like architecture, or graft copolymer, can be developed with pendant peptides as side chain groups on the polymer backbone. 8 X Y + I X-functionalized β-sheet peptide Y-functionalized synthetic polymer β-sheet peptide macroinitiator β-sheet peptide-polymer conjugate "grafting from" (polymerization) "grafting to" (coupling) Figure 1.3: Conjugation approaches to synthesize β-sheet peptide-polymer conjugates 9 No matter how the conjugation is accomplished or which the architecture is, the bioconjugates typically display the characteristic hierarchical fibrous structure organized by the peptide units. A large number of β-sheet peptides have been conjugated to various synthetic polymers with the purpose of building hybrid fibers or hydrogels. Some of the most significant results in the area are reviewed next. The field of polymer-peptide conjugates gained increased attention initially from the pharmaceutical sciences, and lately from materials sciences. When designing a bioconjugate, the selection of an appropriate polymer segment is important as it determines the solubility, and influences the biocompatibility of the overall conjugate system. For pharmaceutical applications, the covalent attachment of a synthetic polymer to therapeutically active proteins leads to reduced biodegradation rates, reduced immune response, reduced toxicity, improved stability by preventing aggregation, and improved solubility [39]. The polymer may also increase the blood circulation time and enhance selective uptake of the conjugate in solid tumors by enhanced permeability and retention (EPR) effect [5]. For materials sciences applications, the attachment of a synthetic polymer to peptides leads to not only structural, but also functional diversity. Stimuli-responsive hybrid conjugates are particularly of interest because of the ability of the polymer to impose responsiveness on the protein/peptide to which it is attached [40]. For example, photosensitive [41] and thermosensitive [42] conjugates were created by covalently attaching photoresponsive polymers, and temperature sensitive polymers, respectively, to various proteins. As the structure of the peptide-polymer conjugate is 1.3.1. Selection of polymer 10 important for determining the function, controlled/living radical polymerization techniques are used to prepare synthetic polymers with well-defined molecular weights and narrow polydispersity indices for immobilization to proteins/peptides. The reversible addition-fragmentation chain transfer polymerization (RAFT) [43, 44] and atom transfer radical polymerization (ATRP) [45] have been used for the synthesis of end-group functionalized polymers that can be directly attached to proteins/peptides without post-polymerization modification. Some of the polymers commonly employed in the synthesis of the peptide-polymer conjugates include poly(ethylene glycol) (PEG) and poly[N-(2- hydroxypropyl)methacrylamide] (poly(HPMA)). PEG conjugation, so called PEGylation, has been widely used to improve the solubility and biocompatibility of peptides and proteins [46]. Many PEGylated therapeutic proteins have been developed and some of them are now in clinical trials or commercially available [5, 47, 48]. Similarly, PEGylation of β-sheet peptides is an effective method for modulating the properties of the self-assembled fibrils, particularly their solubility. Attachment of the linear PEG molecule to a β-sheet peptide retains the hierarchical organization of the fibrils, but reduces their lateral aggregation and controls their width and uniformity [9, 49, 50]. However, although it is the most frequently used polymer, PEG has rather restrained applications because of the limited number of binding sites available for modification with peptides. To overcome this major drawback, other polymers, such as poly(HPMA), are now being evaluated as components of hybrid β- sheet conjugates. 1.3.1.1. Poly(ethylene glycol) 11 Poly(HPMA) is another water soluble, biocompatible polymer that has been extensively used for protein modification in drug delivery applications [51]. Comparing to PEG, the advantage of poly(HPMA) is that it allows attachment to the proteins/peptides via side-chain termini [52]. Although HPMA conjugates and hydrogels have been investigated thoroughly, the self-assembly of copolymers formed from poly(HPMA) conjugated with peptide motifs [53], and in particular with β-sheets, has only recently been evaluated [19, 54]. Just as for PEGylation, peptide β-sheet formation is unaffected by conjugation to poly(HPMA), fibrils formed showing minimal lateral aggregation. In addition, β-sheets in poly(HPMA) conjugates show decreased sensitivity to temperature and pH variations due to the shielding effect exerted by the poly(HPMA) polymer segments. 1.3.1.2. Poly[N-(2-hydroxypropyl)methacrylamide] The concept of peptide-guided assembly of polymers was pioneered in the late 1990 when the first β-sheet-forming peptide derived from an amyloid sequence was attached to PEG [6, 49]. For the first time, it was proven that association of the β-sheets results in a defined peptide core coated at the periphery by the polymer, which acts as a "steric stabilization" layer. Since then, this concept has been verified in numerous other hybrid block copolymers. Conjugation of short β-sheet peptides to PEG in diblock copolymers preserves the self-organization into fibrils and prevents their lateral aggregation [9]. It also provides enhanced stability against variations in concentration and in pH values when compared to native peptide [55]. Similarly, the self-assembly of 1.3.2. Nanofibrous hybrid block copolymers 12 triblock copolymers, in which the β-sheet peptide segment is flanked by two PEG chains, is not affected by pH or temperature changes [14]. However, the hierarchical organization into fibrils typically requires peptides with high propensity for β-sheet formation characterized by strong aggregation tendency and insolubility. Moreover, the length of the polymer and peptide blocks is critical for the nanoassembly of the hybrid copolymer; the longer the peptide segment, the greater the tendency to self-assemble into β-sheets aggregates [56], whereas the longer the polymer segment, the greater the effect on the outcome of the self-assembly [57]. When compared to copolymers containing short polymers, shorter fibers are formed from block copolymers containing a polymer segment with high molecular weight (>5000 Da) [8]. Conjugates may also give rise to nanotubes, fibers, and wormlike or spherical micelles, as the length of the polymer block is increased [57, 58]. An influence of the polymer segment length on the mechanical properties of the fibers has also been observed [56]. These disadvantages can be avoided by using peptides preorganized into templates having an optimized geometry that strongly enhances the formation of antiparallel β- sheets [13, 59]. Hierarchical self-assembly of templated peptides gives rise to helical tapes. Then, by stacking of the tapes, micrometer-sized fiber-like aggregates, with widths controlled by the restricted flexibility impacting the growth of the structure, but with higher hydrophilicity, are formed in the hybrid copolymers. A different approach for overcoming the above mentioned problems has involved the integration of defined structural defects into the peptide blocks [60, 61]. These defects, denoted as "switch" segments, when triggered by adjustments in pH value, are able to disrupt or restore the peptide backbone. As a result, because of the slow changes in the peptide backbone, the 13 self-assembly of the conjugates can be done in a highly controlled manner, leading to well-defined millimeter long fibrils. Although numerous reports have shown that the intrinsic self-assembly ability is retained in hybrid block copolymers obtained by conjugation of various β-sheet peptides with synthetic polymers, research on structure regulation in β-sheet-grafted copolymers has been scarce so far. Few available studies on polyglutamate-grafted polyallylamine [15] and poly(L-leucine) grafted polyallylamine [16] copolymers showed that the formation of amyloid-like fibrils can be easily controlled in graft copolymers by manipulating the pH towards acidic conditions. Conjugation of a short β-sheet peptide as grafts on poly(HPMA) also results in the formation of the fibrils, depending on the concentration [19]. Like for block copolymers, β-sheet peptide grafts have low sensitivity to temperature and pH variations. Moreover, the tendency of the β-sheet peptide to associate into hydrogels is preserved in the poly(HPMA) graft copolymers. 1.3.3. Nanofibrous hybrid graft copolymers Hydrogels are hydrophilic polymer networks that can retain a substantial amount of water while maintaining a distinct 3-D structure. Since 1960's, when Wichterle and Lím proposed the use of poly(2-hydroxyethyl methacrylate) (poly(HEMA)) hydrogels in ophthalmology [62], the applications of these biomaterials have spread, being now used as matrices in tissue engineering, controlled drug release systems, implantable devices, biosensors and actuators, separation systems, nanoreactors, microfluidics and energy 1.3.4. Hybrid hydrogels 14 conversion systems [40, 63]. Such a wide range of applications requires hydrogels with novel properties. Hybrid hydrogels that integrate biological entities with synthetic polymers, interconnected either covalently or non-covalently, were thus created [64, 65]. Enzymes, like glucose oxidase, have been incorporated into pH-sensitive cationic poly(diethylaminoethyl-g-ethylene glycol) hydrogels for controlled delivery of insulin [66]. Enzymes-based hybrid bydrogels, based on adenylate kinase and poly(HPMA), that translate substrate recognition into mechanical motion were also created [67]. Other polymeric gels, have been modified with RGD amino acid sequence to enhance cellular adhesion for constructing scaffolds in tissue engineering [68] or fluorescent DNA for monitoring drug release [69]. Moreover, hydrogels containing covalently attached growth factors have been created. Transforming growth factor beta (TGF-β) was bound to PEG to regulate smooth muscle cell function [70], whereas bone morphogenic protein-2 (BMP-2) was attached to alginate to facilitate osteoblast differentiation inside gels [71]. Unprecedented levels of structural organization in hybrid hydrogels are also required. As a result, physical gels, or self-assembling gels, which are polymer networks held together through secondary molecular interactions between protein/peptide segments, have been lately developed. For their formation, recognition motifs found in Nature, such as coiled-coils [53, 72-74], β-sheets [19, 26, 75-77], complementary oligonucleotides [78], or antigen-antibody interaction [79], are used. The high degree of control over the properties of these hydrogels is typically the result of the responsiveness of the protein/peptide crosslinks directly related to the molecular recognition. Although the concept is not new, the possibility to create hybrid hydrogels using protein folding modules as physical crosslinkers has not been fully exploited so far. Such 15 hybrid hydrogels were first assembled from poly(HPMA) copolymer and coiled-coil-forming peptides through metal complexation [72]. These hydrogels were able to undergo significant volume transitions at the melting temperature of the coiled-coil modules. For the first time, it was thus shown that the behavior of the coiled-coil domains can be imposed on the self-assembled hybrid hydrogels. In order to avoid the disadvantage of this first design, which is the association of the grafts attached on the same molecule into dimers, and to guarantee the formation of a well-defined 3-D structure, graft poly(HPMA) copolymers with complementary, oppositely charged antiparallel coiled-coil heterodimer grafts can be employed [53, 74]. Enhanced homogeneity due to a decreased steric hindrance due to the "in register" alignment of the peptides is achieved; however, a threshold amount of grafts per polymer chain is needed for hydrogel formation. The concentration of the graft copolymers also has an important impact on the kinetics of gelation, as well as on the structure of the hydrogels. The first hybrid hydrogel crosslinked with β-sheets was reported in 2000, by Kopeček and Stewart [17]. It was a polyacrylamide-based hydrogel crosslinked with genetically engineered single immunoglobulin (Ig)-like domain from the human cardiac titin, an elastic muscle protein that contains antiparallel β-sheets. Not only that the β- sheet protein modules were able to control the structure, but they also mechanically modulated the response of the hybrid hydrogel to temperature, ionic strength, as well as swelling, in a predictable manner. Therefore, it was shown for the first time that properties of a well-defined β-sheet protein motif can be imposed onto a hybrid hydrogel mainly formed of synthetic polymer chains. 16 Another step was made by Kopeček's laboratory when, following the model of coiled-coil hybrid hydrogels [53, 74], a novel hybrid hydrogel whose formation is mediated by β-sheet motifs was synthesized and characterized [19]. As for coiled-coil hybrid hydrogels, a certain number of grafts per macromolecule are needed for gelation. This physically crosslinked hydrogel forms spontaneously under appropriate concentration and incubation time. Its morphology is characterized by long-range order with uniformly aligned lamellae, beneficial in directing mineral deposition and cellular migration, which makes the β-sheet hybrid hydrogels appealing for applications as scaffolds in bone tissue engineering. Biomineralization is the process by which biological systems produce proteins that self-assemble into matrices able to control the nucleation, growth and termination of mineral phases [80]. It is of particular interest to researchers for studying bone tissue growth and regeneration, as well as for developing biomimetic materials. In Nature, specific proteins were shown to serve as scaffolds for controlling the nucleation and growth of various inorganics. In fact, biomineralization in bone has been linked to acidic proteins in β-sheet conformation [80, 81]. Similarly, β-sheet peptides self-assembled into fibers are able to direct mineralization of hydroxyapatite (HA), to form a composite material in which the crystallographic c axes of HA are aligned with the long axes of the fibers [81], as shown in Figure 1.4. This alignment of the crystallographic c axis along the long axis of the fibers is the same as that observed between collagen and HA crystals in bone. 1.4. Biomineralization in β-sheet self-assembled materials 17 c axis of HA crystals long axis of collagen/β-sheet fibers Figure 1.4: Alignment of the HA crystals with collagen/β-sheet fibers (modified after [81]) 18 HA is the calcium phosphate mineral found in vertebrate bones and teeth. It belongs to the apatite family, minerals with the chemical formula Ca10(PO4)6X2, where X=OH- in derivates of hydroxyapatite or X=F- in fluoroapatite [82]. Given the involvement of different proteins in the mechanism of apatite crystal formation, biogenic apatite is different from the geological one with respect to size, crystallinity, and structure organization. Based on X-ray diffraction patterns, bone HA formula was identified as being Ca5(PO4)3OH, with a Ca/P ratio of 1.67 [83]. This value can vary significantly due to carbonate substitutions, OH- deficiencies, and imperfections in the crystal lattice that give rise to different calcium phosphate minerals. Biologically relevant calcium phosphate minerals are shown in Table 1.1. 1.4.1. Hydroxyapatite Bone is a complex, highly organized and specialized tissue made of a composite of 65-70% mineral (mostly nanoscale HA crystals) and 25-30% organics (collagen, glycoproteins, proteoglycans and sialoproteins) [87]. Its architecture consists of several hierarchical levels. The first level includes the molecular components among which small HA crystals (30-50 nm long, 20-25 nm wide, and 1.5-4 nm thick) [82]. These crystals are plate-shaped, although HA needles have been also observed [88]. The second level is formed by the mineralized collagen fibrils having HA crystals with c axis aligned with their long axis. Mineralized fibrils form arrays constituting the third level of organization. Other levels of hierarchy are made of osteons - structures involved in bone remodeling, either spongy (trabecular) or compact (cortical) bone, and simply the macroscopic bone. 1.4.2. Bone mineralization 19 Table 1.1: Biological apatite minerals mineral chemical formula Ca/P ratio references monocalcium phosphate monohydrate (MCPM) Ca(H2PO4)2•H2O 0.5 [82, 84] dicalcium phosphate dihydrate (DCPD) (brushite) CaHPO4•H2O 1.0 [82, 84] octacalcium phosphate (OCP) Ca8H2(PO4)6•5H2O 1.33 [82, 85] amorphous calcium phosphate (ACP) Ca3(PO4)2•nH2O 1.5 [82] tricalcium phosphate (TCP) Ca3(PO4)2 1.5 [85, 86] HA Ca10(PO4)6(OH)2 1.67 [82, 83, 85] carbonated HA (dahllite) Ca10(PO4,CO3)6(OH)2 1.67 [82] fluoroapatite Ca10(PO4)6F2 1.67 [82, 86] tetracalcium phosphate (TTCP) Ca4(PO4)2O 2 [86] 20 Depending on the type, bone forms by two processes: by intramembranous ossification (cranial bones) involving direct differentiation of mesenchymal progenitor into osteoblasts, or by endochondral ossification (the rest of the skeleton) involving mineralization of a cartilaginous template [82]. The mineralization process starts with the apoptosis of the chondrocytes and the migration of the osteoblast cells into the template. The new bone is initially deposited by osteoblasts as woven bone containing a high proportion of osteocytes and disorganized collagen fibers. During development or remodeling, this woven bone is slowly replaced with a stronger, highly ordered lamellar bone [82]. While there is no consensus regarding the HA formation in vivo, it is believed that the initial mineral formation is under cellular control, whereas the mineral propagation, crystals nucleation, growth and orientation are mediated by the collagen fibers and/or matrix proteins. One can say that biomineralization, as a process, is a form of natural self-assembly similar to the one responsible for β-sheet peptides organization into hierarchical levels. The earliest apatite nuclei are irregularly shaped, thin dots, which, in time, become long platelets. The growth is also accompanied by an increase in crystallinity and presumably, by a phase transformation from intermediary calcium phosphate apatites to HA [89]. In reality, bone capacity for healing and remodeling is finite. A series of medical problems caused by bone mineralization related diseases, among which osteoporosis and osteomalacia, led to the development of new strategies to repair and regenerate this tissue. While bone grafts and collagen scaffolds are still widely used, their associated problems like disease transmission, immunological rejection, poor incorporation, and batch to batch variation, immunogenicity, complex molecular structure, poor mechanical 21 strength, respectively, shifted the gear towards synthetic systems based on self-assembled nanofibrous peptides and hybrid materials. Bone mineralization is a process naturally induced in collagenous and non-collagenous proteins that provide appropriate conditions for nucleation and growth of ordered crystals of HA. Such proteins contain β-sheet motifs with negatively charged groups that have been used for the design of self-assembling fibrous peptides able to promote the formation of HA crystals by attracting Ca2+ [90]. Moreover, they present planar surfaces in which the distance between two phosphorylated serine residues is identical to the distance between two Ca2+ atoms in HA (6.9 Å). Therefore, it is believed biomineralization in these proteins is the result of epitaxy that favors HA crystal nucleation and orientation. One example is enamelin, the major acidic protein of tooth enamel, which has been shown to adopt a β-sheet conformation during enamel formation [91, 92]. 1.4.3. Biomineralization in β-sheet nanofibers The intriguing structural correspondence between the dimensions of the hexagonal unit cell in HA crystal (a=b=9.432 Å, c=6.881 Å) and the dimensions of the repeat units in self-assembled β-sheets (distance between any two β-strands=4.8 Å, about half of a or b axes of the apatite unit cell, whereas distance between every second amino acid along a β-strand=6.9 Å≈c), once again suggests their involvement in apatite crystallization [91]. As a result, many β-sheet peptides self-assembled into fibrils are used as templates for nucleation of HA. For example, designed peptide-amphiphile (PA) structures in acidic conditions at pH 4 are able to self-assemble into fibers that template 22 the mineralization of the HA from a solution of CaCl2 and Na2HPO4, in a way that mimics the alignment between collagen fibrils and apatite crystals in natural bone [81]. Mineralization can be also induced at different pH values by simple modifications of the peptide length and amino acid composition that promote self-assembly, and respectively nucleation of HA [93]. Furthermore, biomimetic synthesis of HA in vitro can be accomplished by mimicking biomineralization in simulated body fluid (SBF). SBF is a solution with ion concentrations nearly equal to those of human blood plasma, but highly supersaturated with respect to apatite [94]. To achieve controlled nucleation and oriented growth of HA crystals, β-sheet fibers are incubated in SBF for up to 4 weeks. Although the biomineralization mechanism has not been completely elucidated, it is believed that anionic groups on the surface of the fibers, such as -COOH and -OH, which act as proton acceptor and proton donor, respectively, first attract HPO4 2- from the solution forming a hydrogen-bonded complex - the nucleation site [95]. This complex then binds inorganic Ca2+ as a consequence of the electrostatic effects and undergoes a phase transition, which eventually leads to nucleation and growth of HA crystals (Figure 1.5). In the process, the self-assembled β-sheets are also responsible for inducing the domain ordering in the HA crystals [97]. Crystals thus formed have c axes aligned with the longitudinal axes of β-sheet fibers. Peptide-based mineralization systems can be considered basic models for HA mineralization since their features closely mimic the organization of collagen fibers in natural bone. 23 HPO4 2- OH- Ca2+ SBF C O O-C O O-C O O-C O O-C O O-C O O-HA nucleus SBF Figure 1.5: Mechanism of HA nucleation on the surface of self-assembled β-sheets incubated in SBF (modified after [96]) 24 Despite the fact that HA has been largely used as coatings to enhance osteointegration of metallic and ceramic implants, only in the peptide-based mineralization systems, the level of nano and macro organization and the organic-inorganic integration seen in real bone can be achieved. Therefore, there is an acute necessity of expanding the constructs in which biomineralization process occurs from simple to more complex architectures, like hydrogels. In order to act as a scaffold for bone growth, a hydrogel not only should serve as a reservoir for water and nutrients, but should also have some essential properties: biocompatibility, porosity (open, interconnected pores with sizes within the 100-900 μm range), mechanical properties that ideally match those of living bone, and osteoconductive and osteoinductive abilities [87, 98]. In an effort to achieve these requirements, bone scaffolds based on biopolymeric materials like collagen [99, 100], chitosan [101], alginate [102], cellulose [103], and silk [104] gels were developed. Organic hydrogel systems have been also used for the fabrication of bone-like composite materials by exploiting the affinity of the calcium apatite to the anionic polymeric surfaces of poly(HEMA) [105, 106], or poly(vinyl alcohol) (PVA)/poly(acrylic acid) (PAA) complex [95]. Although these biomimetic systems are relatively simple and provide materials with good mechanical properties, the static preparative conditions they use result in a prolonged time to reach complete HA formation (up to 2 months). Attempts to faster prepare HA in hydrogels have included electrophoresis [107], or conjugation with RGD and BMP [108]. 1.4.4. Biomineralization in hydrogels 25 On the other hand, as discussed in previous sections, a mimetic material can be obtained only when the level of HA organization encountered in natural bone is reproduced. Most of the systems presented above fail to do that. However, given its importance in the formation of bone, collagen became an obvious scaffold on which to study the synthetic mineralization of HA. On the basis of the c axis preferential alignment of the mineral with longitudinal axes of the fibrils, collagen acts as a template for HA precipitation. Despite that, disadvantages of collagen, related mainly to poor availability and immunogenicity, prevent its use in some bone tissue engineering applications. Therefore, it is needed to develop new materials that provide the scaffolding properties needed for the intended application. Nanofibrous peptides, designed to mimic properties of collagen, are now used to create hydrogels for this purpose. For example, peptides with β-hairpin structure able to self-assemble into fibers, when placed in saline solution [109] or exposed to light [110], form hydrogels that were found to support cell adhesion and migration. These hydrogels have been investigated so far as scaffolds for cartilage regeneration, but good mechanical properties make them interesting candidates for bone tissue engineering as well. Fibrillar networks self-assembled from oligomeric β-sheet-forming anionic peptides have been capable of nucleating apatite mineral crystals after incubation in SBF for 7 days at 37 oC, and were used as scaffolds for dental tissue [111, 112] or bone tissue regeneration [113]. 3-D networks formed of "mineral directing gelator" β-hairpin peptides were also found to direct the mineralization process of calcium and phosphate to produce HA [114]. The early success of fibrillizing peptide materials in biomineralization is promising for clinical use, but improvements need to be made with respect to the 26 structure and properties of the scaffolds [115, 116]. A hybrid multicomponent hydrogel that would reproduce the structure of bone extracellular matrix (ECM) and control the deposition and oriented growth of HA crystals is likely the most encouraging approach for scaffold-based bone tissue engineering. Such a hybrid hydrogel system would be obtained from noncovalent interactions established between self-assembled fibers formed by conjugates of β-sheet peptides and synthetic polymers. Moreover, once biomineralization would take place, biomimetic apatite-coated β-sheet fibers, components of the hybrid hydrogel, would be able to show better cellular response represented by better proliferation of osteoblasts and increased osteogenic markers expression [117]. Currently, research in bone tissue engineering is aiming at creating living tissue constructs that are functionally, structurally and mechanically comparable to the natural bone. For this purpose, scaffolds that support bone cells colonization, migration, growth and differentiation, and have specific characteristics including high porosity, high surface area, mechanical strength and specific 3-D shape are needed. Scaffolds made from hybrid hydrogels potentially possess these required characteristics for applications in 3-D tissue culture and regenerative medicine. They are able to bridge the gap between natural and synthetic scaffolds by combining well-characterized synthetic materials with functionally relevant proteins/peptides. 1.5. Hybrid hydrogels for bone tissue engineering The formation of bone tissue analogs requires a cell supporting matrix with properties, structure, and composition similar to bone ECM [118]. A 3-D hybrid scaffold that mimics the properties of the bone ECM is critical to preserve the cells ability to 27 differentiate. For an optimal function, the bone hybrid scaffold should posses a series of properties including biocompatibility, osteoconduction and osteoinduction, mechanical support, and adequate water and nutrients flow [119, 120]. Controlling the structure at nano and macro level is also essential for the regenerative capacities of the hybrid gel. A highly porous surface with interconnected pores and a high internal surface area to volume ratio are desired for cell adhesion, growth and migration, as well as for diffusion of nutrients and metabolic waste products [121]. Some studies on bone regeneration indicate the need for pore size ranging from 100 to 500 μm [122-125], while others successfully employed scaffolds with a pore size equal to the size of the cell used in the application [126]. Special attention should be given to the porosity in the design since this factor is critical in determining the mechanical properties of the scaffold; increasing the porosity, significantly decreases the strength of the matrix. Improved artificial hybrid scaffolds, in which the porosity is strictly controlled, can be generated through physical crosslinking such as hydrogen bonding by using protein folding motifs and protein-protein interactions [127]. β-Sheet motif is particularly of interest due to its ability to self-assemble into fibers that mimic the characteristics of natural ECM. Hydrogels self-assembled from β-sheets are highly attractive materials for developing synthetic ECM analogs to enhance bone regeneration, as they have controlled porosity, nanostructure, and mechanical properties matching those of bone ECM. In fact, microporous silk fibroin (a protein with mostly β-sheet structure) scaffolds have been already fabricated by electrospinning technique and characterized. Aligned electrospun fibroin fibers are able to guide the orientation of bone marrow-derived mesenchymal stem cells [128]. Moreover, adhesion and spreading of the 28 cells, and mineralization of the matrix can be enhanced through incorporation of adhesion ligands, specifically arginine-glycine-aspartic acid (RGD) [129], and BMP-2, respectively [130]. The employment of RGD cell-binding sequence is the most advantageous to induce fibroblasts and osteoblasts attachment and spreading in a concentration-dependent manner [131-134]. The number of attached cells as well as their function is directly related to the RGD surface density; attachment of the cells and expression of bone-related markers osteocalcin (OCN) and alkaline phosphatase (ALP) occur only above a RGD threshold concentration of 2.5 mM [135]. Ligand RGD spacing and island distribution within the hydrogel scaffold are equally important for the osteoblasts adhesion, proliferation and differentiation [136, 137]. With respect to macrostructure, it should be taken into account that cells behave differently when cultured on 2-D and 3-D platforms. Differences in concentration of nutrients, differentiation, proliferation and apoptosis, as well as migration of cells exist between a 2-D surface and a 3-D environment [138]. For example, human breast epithelial cells develop like tumor cells when cultured in 2-D, but show normal growth behavior when cultured in a 3-D gel [139]. Also, cell morphology can be significantly different; usually in a 3-D culture the cells tend to retain their rounded morphology [140, 141], because of the dramatically altered cell integrin expression as compared to 2-D culture [142]. Moreover, in 3-D culture, cell binding to the hydrogel scaffold is needed, as it is thought to provide the signals for long-term viability and functionality, and suppressed apoptosis. In the absence of cell-matrix interactions, anchorage-dependent cells undergo anoikis, a form of apoptosis that occurs when cells detach from the ECM [143, 144]. Luckily, direct cell adhesion of the pre-osteoblasts to the 3-D scaffold can be 29 achieved through incorporation of integrin-binding RGD peptide [145, 146]. Using 3-D fibrous scaffold architecture for bone tissue engineering applications proves to be advantageous in this respect; observed biological effects include higher osteoblastic progenitor cells (MC3T3-E1) attachment [147], enhanced osteoblastic marker genes' expression indicating differentiation of the cells [148], and significant mineral deposition [149]. The composition of bone ECM, which is a type of organic-inorganic nanocomposite with HA crystals organized at nanoscale, is another factor that should be simulated by the hybrid hydrogel scaffold in order to achieve a good biomechanical function [150]. Combining nanofibrous materials with pre-synthesized inorganic materials, by simple blend-mixing, is one way to generate bone scaffolds with improved osteogenic differentiation and calcification, as well as mechanical properties [151-154]. However, due to the structure and size difference comparing to bone mineral crystals, synthetic calcium phosphates have long degradation times in physiological environment, which limits their application in bone tissue engineering [155]. Another way is to form the inorganic material in situ through activation of the nanofibers in an alkaline solution to generate carboxylic groups [156], followed by mineralization in SBF [157, 158]. The β-sheet fibrous substratum promotes the deposition of calcium phosphate minerals and provides favorable conditions for controlled mineralization, and more importantly, supports the bone-forming cell culture [159], therefore is presumably a suitable bone matrix. The processes by which the bone scaffolds are manufactured should provide high control over the structure and composition in order to yield the required properties: 30 accurate 3-D geometry, and interconnected pores with regular morphology, size and distribution. Several techniques have been developed and employed for bone scaffolds fabrication including solvent casting, particle leaching, freeze drying, phase separation, self-assembly, electrospinning, and melt molding [118, 121]. Self-assembly and electrospinning are particularly attractive because they produce fibrous materials similar to ECM, whereas particle leaching, freeze drying and phase separation provide tailored porosity and pore structure [121, 150]. A combination of these techniques is recommended for strict control over the structure and properties of the scaffold. The objective of this project is to develop novel hybrid biomaterials that can self-assemble into hydrogels with controlled structural features at the nanoscale through interactions among β-sheet-peptide-based nanofibers. To achieve this goal, we attempted to identify the relationship between structure and function, by designing and synthesizing hybrid nanofibrous materials with spatial organization provided by specific folding, and by employing them as bioactive scaffolds that promote osteoblasts attachment, proliferation, and differentiation. Precisely, we combined self-assembling β-sheet peptide sequences with hydrophilic poly(HPMA) to produce customized nanoscale engineered biomaterials for applications in bone tissue engineering. There are two major hypotheses: 1.6. Statement of objectives 1) A β-sheet peptide is able to impose its structural arrangement on the overall structure of a conjugate; conjugation of a β-sheet peptide as blocks or grafts with poly(HPMA) could result in the formation of a fibril-like nanostructure for the copolymer and, with increasing concentration, of a hybrid hydrogel. 31 2) A hybrid hydrogel self-assembled from copolymers of poly(HPMA) and β-sheet peptides could mimic the natural bone ECM and provide surfaces characterized by epitaxy that favor HA crystals nucleation and oriented growth, as well as support for bone cells. To test these hypotheses, the following specific aims were proposed: 1) To design and synthesize hybrid diblock copolymers, based on poly(HPMA) and β-sheet peptide blocks, and evaluate their self-assembly into nanofibrous materials. 2) To design and synthesize hybrid hydrogels, based on poly(HPMA) grafted with β- sheet peptides, and characterize their self-assembled structure at nano and micro scales. 3) To design, synthesize and characterize a novel bone scaffold based on a hybrid hydrogel self-assembled from graft copolymers of poly(HPMA) and complementary β-sheet peptides. Over the past years, our laboratory has designed, synthesized and characterized self-assembling gels based on coiled-coil folding motif. The development of a hybrid hydrogel self-assembled from β-sheet domains is the consequence of a necessity: better control over the gel architecture in order to emulate the structure and the function of natural ECM. 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Silberstein, Hydrogel scaffolds of amphiphilic and acidic β-sheet peptides. Adv. Funct. Mater. 18 (2008) 2889-2896. 44 NOTE: This chapter was reprinted with permission from the following publication: L. C. Radu, J. Yang, J. Kopeček, Self-asembling diblock copolymers of poly[N-(2-hydroxypropyl)methacrylamide] and a β-sheet peptide. Macromol. Biosci. 9 (2009) 36-44. CHAPTER 2 SELF-ASSEMBLING DIBLOCK COPOLYMERS OF POLY(HPMA) AND A β-SHEET PEPTIDE Lately, there is an increasing interest in developing peptide-polymer hybrid materials that combine the advantages of individual components. Herein, the self-assembly of hybrid diblock copolymers, composed of poly(HPMA) and β-sheet peptide (P11; CH3CO-QQRFQWQFEQQ-NH2) blocks, was investigated. Copolymers were synthesized via thiol-maleimide coupling reaction, by conjugation of semitelechelic poly(HPMA)-SH with maleimide-modified β-sheet peptide. As expected, circular dichroism and Congo Red binding studies showed that the peptide block imposed its β- sheet structural arrangement on the structure of diblock copolymers. Transmission electron microscopy and atomic force microscopy proved that the peptide and these copolymers had the ability to self-assemble into fibrils. 2.1. Summary 46 Molecular self-assembly of synthetic block copolymers has been lately the focus of research activity of numerous investigators [1-3]. While synthetic copolymers are still of interest, the possibility to prepare hybrid block copolymers based on combination of proteins or peptides and synthetic polymers has become an attractive way to expand the diversity of structures and functions in synthetic materials. Peptide/protein-polymer hybrid copolymers are able to overcome some of the disadvantages of the individual components. For example, a structural protein or peptide block (such as coiled-coil or β- sheet) may control the nanostructure of the synthetic component [4-6], whereas a synthetic polymer block may increase the stability [7] and the biocompatibility of the protein/peptide segment [8, 9]. 2.2. Introduction A series of interesting reports has described the synthesis, as well as the self-assembly of different β-sheet peptide-PEG hybrid diblock copolymers [10-15]. Two main approaches to obtain hybrid copolymers can be identified: polymerization strategies, using a peptide macroinitiator to synthesize the polymer block, and coupling strategies, using a preformed synthetic polymer to react with selective functionalities on the peptide [16, 17]. However, when analyzing the self-assembly mechanism, a common trend rises: the hierarchical organization of the β-sheet motifs in the peptide blocks mediates the self-organization of the conjugates. As a result, fibrils can be formed by stacking of the β- sheets on top of each other [18]. Studies showed not only that the self-organization of different β-sheet peptides into fibrils was preserved after conjugation with PEG, but moreover, the lateral aggregation of these fibrils was prevented [10, 14]. It seems that the PEG coating provides solubility and facilitates self-assembly into uniform fibrils with no 47 lateral aggregation. This is particularly important in case of PEG-coated amyloid fibrils that exhibit a strong reduction in the formation of amyloid plaques [14], associated with, e. g., Alzheimer's disease. While various β-sheet peptides have been successfully conjugated with PEG, there is no report on their conjugation with poly(HPMA), a nonimmunogenic, neutral, hydrophilic polymer currently employed in the delivery of anticancer drugs. Compared to PEG conjugates, imposing a certain secondary structure on a poly(HPMA)-peptide conjugate might be a challenging task, since in water, poly(HPMA) has a random coil conformation, while PEG adopts an extended conformation [19]. However, previous work on HPMA copolymers containing coiled-coil peptide grafts demonstrated that self-assembly of the hybrid poly(HPMA)-peptide copolymer systems is possible, and moreover, it can even trigger the formation of a hydrogel [20, 21]. In this article, the synthesis and the self-assembly of hybrid diblock copolymers, based on poly(HPMA) and β-sheet peptide blocks are described. We hypothesized that the β-sheet-forming peptide block will impose its structural arrangement on the overall structure of the diblock copolymers as illustrated in Figure 2.1. The β-sheet peptide was synthesized using solid-phase methodology and manual Fmoc/tBu strategy, and purified by reverse phase (RP) HPLC. The sequence (P11-2; CH3CO-Gln-Gln-Arg-Phe-Gln-Trp- Gln-Phe-Glu-Gln-Gln-NH2) [22] (herein termed P11), previously designed by Aggeli et al., was used. We chose this well-characterized, short peptide sequence for the fact that in aqueous solutions, it can form antiparallel β-sheets self-organized into fibrils [23], as well as for the easiness of its synthesis. 48 Figure 2.1: The hypothesized self-assembly of β-sheet-poly(HPMA) diblock copolymer 49 A novel approach to prepare the hybrid diblock copolymers was developed. Well-defined semitelechelic poly(HPMA) polymers terminated with thiol functional groups were prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization in methanol followed by aminolysis. Then, the N-terminus of the P11 peptide was modified with a maleimide group, and finally, in the last step, each conjugation was accomplished via the maleimide-thiol coupling reaction. Circular dichroism spectroscopy (CD) and Congo Red (CR) binding studies were performed to characterize the secondary structure of the peptide and of the peptide domain in diblock copolymers. The morphology of the materials was investigated by transmission electron microscopy (TEM), and atomic force microscopy (AFM). 2.3. Experimental part Side chain-protected Fmoc-amino acids, rink amide MBHA resin were from Novabiochem (San Diego, CA). 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3- tetramethyluronium hexafluorophosphate (HATU; >98%) and 1-hydroxybenzotriazole (HOBt) were purchased from AK Scientific (Mountain View, CA). N,N-dimethylformamide (DMF; 99.8%), sodium hydrosulfite (Na2S2O4; 79%), piperidine (99.5+%, Biotech grade), triisopropylsilane (TIS; 99%), N,N-diisopropylethylamine (DIPEA; 99%), acetic anhydride (99+%) and methanol were from Sigma-Aldrich (St. Louis, MO). Ethyl ether and dichloromethane (DCM) were from Maleinckrodt Baker (Philipsburg, NJ). Trifluoroacetic acid (TFA; 99%) was purchased from Acros Organics (Morris Plains, NJ). Succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate 2.3.1. Materials 50 (SMCC) was from Soltech Ventures (Beverly, MA). 4,4'-Azobis(4-cyanopentanoic acid) (V-501), N,N'-diisopropylcarbodiimide (DIC; >98%), hexylamine (>98%), and tris(2- carboxyethyl)phosphine (TCEP) were from Fluka (Milwaukee, WI). Thiol modified poly(ethylene glycol) (CH3-PEG-SH; weight average molecular weight, Mw, 1892 g/mol; polydispersity, Mw/Mn, 1.21; SH amount, 498 μmol/g) was purchased from Rapp Polymere (Tübingen, Germany). Congo Red (CR; dye conte≈nt 80%) was from Alfa Aesar (Ward Hill, MA). The chain transfer agent (CTA), 4-cyanopentanoic acid dithiobenzoate [24], and HPMA [25] were synthesized as previously described. P11 peptide, CH3CO-Gln-Gln-Arg-Phe-Gln-Trp-Gln-Phe-Glu-Gln-Gln-NH2 [22], was synthesized using solid-phase methodology and manual Fmoc/tBu strategy on rink amide resin, similar to a previously described protocol [4]. After swelling of the resin beads (200 mg, 0.14 mmol) in DCM (5 mL), and deprotection in 20% piperidine in DMF (2.5 mL, 2×5 min), the first amino acid, Fmoc-Gln(Trt)-OH (0.35 mmol), was attached to the resin in DMF, in the presence of HOBt and DIC. The rest of the amino acids (each 0.175 mmol) were dissolved in DMF/HOBt solution and attached to the resin-bound peptide in DMF after deprotection, one at the time, in the presence of HATU and DIPEA (each 0.175 mmol). The completion of each coupling step was verified by Kaiser test. The final 11th residue was deprotected, and the peptide-resin was washed with DMF, DCM, and methanol, and dried under vacuum. Part of the resin-bound peptide was treated with a solution of acetic anhydride and DIPEA in DCM for acetylation of N-terminus, and then, with TFA/TIS/H2O cocktail solution (95:2.5:2.5, vol.-%) for cleavage 2.3.2. Peptide synthesis and purification 51 of the peptide from the resin beads and deprotection of the side chains. Crude P11 peptide was precipitated with ethyl ether. The rest of the resin-bound peptide was kept in DMF and further used for coupling with semitelechelics poly(HPMA) and PEG. Peptide purification was carried out by RP-HPLC using a semipreparative Zorbax 300SB-CN column (9.4×250 mm, 5 μm particle size, 300 Å pore size) from Agilent Technologies. The peptide was eluted with a linear gradient at a flow rate of 2 mL/min, using buffer A, H2O with 0.1% TFA, and buffer B, acetonitrile with 0.1% TFA. The purity of the collected fractions was verified with analytical RP-HPLC, using an Eclipse XDB-C8 column (4.6×150 mm, 5 μm particle size, 80 Å pore size). Collected fractions were lyophilized. The identity of the P11 peptide obtained after lyophilization was confirmed by MALDI-TOF mass spectrometry (MS; Voyager-DE STR Biospectrometry Workstation, PerSeptive Biosystems, Framingham, MA), showing a single main peak corresponding to the expected molecular weight at M++1, 1593.76 m/z. 2.3.3. Synthesis and purification of β-sheet peptide-poly(HPMA) Two RAFT polymerizations of HPMA were performed following a procedure described by McCormick and co-workers [26], employing 4,4'-azobis(4-cyanopentanoic acid; V-501) as initiator, and 4-cyanopentanoic acid dithiobenzoate as chain transfer agent (CTA). HPMA polymerizations were conducted at 60 oC in methanol, using initial monomer concentration ([Mo]) as 1 M, and constant ratio of CTA to initiator as 5, in sealed ampoules that were purged with nitrogen for 30 min prior to reaction. Monomer to CTA ratios ([Mo]/[CTA]) of 25 and 40 were used. Polymerization reactions were diblock copolymers 52 allowed to proceed for 24 h. The resulting polymers were precipitated into cold ethyl ether and washed with acetone for removal of the unreacted HPMA monomer. After freeze-drying of the products, the polymerization yields were estimated as being 48.5% and 98.4%, respectively. The molecular weights of poly(HPMA) were analyzed by size exclusion chromatography (SEC) on an Äkta FPLC system (Amersham Pharmacia Biotech) equipped with UV and RI detectors, using a Superdex Peptide column, previously calibrated with HPMA fractions, and phosphate buffer solution (PBS, pH 7.2) as eluent at a flow rate of 0.4 mL/min. MALDI-TOF mass spectrometry was also employed for the evaluation of the polymers molecular weight. Both techniques confirmed that two low-molecular weight poly(HPMA) polymers (poly(HPMA)2k with a weight average molecular weight, Mw, of 1990 g/mol and a polydispersity, Mw/Mn, of 1.41, and poly(HPMA)5k with a Mw of 4890 g/mol and a Mw/Mn of 1.14) were synthesized. HPLC experiments and MALDI-TOF mass spectrometry showed that P11 peptide even in crude form was pure (~85%), which enabled the use of the peptide-bound resin for the synthesis of the diblock copolymers. For each coupling, part of the peptide-bound resin batch (0.046 mmol, 72 mg) was modified with maleimide group. A solution of SMCC (0.07 mmol, 23.4 mg) and DIPEA (0.138 mmol, 24.4 μL) was added to the peptide-bound resin beads in 2 mL DMF and kept at room temperature for 20 h. The identity of the maleimido-modified P11 peptide was verified by MALDI-TOF mass spectrometry, after a small amount of the product was cleaved from the resin by using TFA/TIS/H2O cocktail solution (95:2.5:2.5, vol.-%), precipitated with cold ethyl ether, 53 isolated by filtration, dried, dissolved in DI water, and lyophilized. A single peak corresponding to the expected molecular weight at M+, 1770.84 m/z, was obtained. The thiocarbonate end groups of poly(HPMA)2k (160 mg, Mw=1990 g/mol, Mw/Mn=1.41) and poly(HPMA)5k (160 mg, Mw=4890 g/mol, Mw/Mn=1.14) were transformed into thiol groups by aminolysis [27]. Each polymer was dissolved in 2 mL DMF and bubbled with nitrogen for 30 min. Aqueous Na2S2O4 solution (0.5 M, 50 μL) and hexylamine (0.1 g, 1.0 mmol) were added to the mixture. The reaction was allowed to proceed for 18 h at room temperature, under nitrogen atmosphere and magnetic stirring. Discoloration of the solutions was observed. The polymers were precipitated into ethyl ether, isolated by centrifugation, dried, dissolved in DI water, and lyophilized. Ellman test was performed on the thiol-terminated semitelechelic poly(HPMA) for quantification of thiol groups [28] as being 0.57 mmol/g for poly(HPMA)2k, and 0.22 mmol/g for poly(HPMA)5k, respectively. Thiol-terminated poly(HPMA)2k and 5k (130 mg each) were incubated in DMF in the presence of TCEP (4.8 mg) for 2 h, to eliminate the formation of the polymeric disulfides [29]. The maleimide-modified P11 peptide bound to the resin beads and the corresponding thiol-modified poly(HPMA) were mixed in 2 mL DMF. The maleimide-thiol coupling reactions took place in 24 h at room temperature. Following coupling, the products were cleaved from the beads by using a TFA/TIS/H2O cocktail solution (95:2.5:2.5, vol.-%), precipitated with ethyl ether, collected by filtration, dried in air, dissolved in DI water, and lyophilized. HPLC analysis of the products showed that a mixture of free P11 peptide and P11-poly(HPMA) conjugate was obtained after each coupling reaction. The conjugates were purified by dialysis (molecular weight cutoff, 54 MWCO, 1000 Da) against water for 72 h. Analytical RP-HPLC confirmed that pure peptide-polymer conjugates (P11-poly(HPMA)2k and P11-poly(HPMA)5k) were obtained after dialysis. The yield of the coupling reaction was calculated as being 8% for P11-poly(HPMA)2k, and 6.66% for P11-poly(HPMA)5k with respect to the pure conjugates. For comparison, a similar coupling approach was employed for the conjugation of thiol-modified PEG (Mw=1892 g/mol, Mw/Mn=1.21, SH content=0.49 mmol/g) with the maleimide-modified P11 peptide bound to the resin beads. The yield of the reaction was 14.5% with respect to the pure P11-PEG2k diblock copolymer. Circular dichroism (CD) spectra were collected on an Aviv 62DS CD spectrometer at 25 oC. The samples were prepared by dissolving a known mass of lyophilized product in water. The pH of the solutions was adjusted under pH-meter monitoring (Corning pH-meter 440) to the desired values (2, 7 or 11) by addition of appropriate amounts of 1 N HCl or 1 N NaOH solution, unless otherwise stated. Solutions with concentrations determined by UV spectroscopy (typically, 1 mg/mL, 0.6 mM, with respect to the peptide) were incubated at room temperature for 3-10 days or longer prior to CD measurements. The pH of the solutions was checked periodically and adjusted when required. Wavelength scans were recorded at 1 nm intervals from 250 nm to 200 nm using a 0.1 cm path length quartz cuvette. Spectra were averaged from three consecutive scans and subtracted from the background. Ellipticity measurement was shown to mean residue ellipticity ([θ], in deg·cm2·dmol-1) and calculated as (Equation (1)) [21] 2.3.4. Circular dichroism spectroscopy 55 [θ] = [θ]obs (MRW / 10 l c), (1) where: [θ]obs is the ellipticity measured in millidegrees, MRW is the mean residue molecular weight of the peptide (molecular weight of the unacetylated peptide, 1551 Da, divided by the number of amino acid residues), l is the optical path length of the cell in cm (0.1 cm), and c is the peptide concentration in mg·mL-1. Spectrophotometric analysis of CR binding to P11 and to P11-poly(HPMA)2k and P11-poly(HPMA)5k diblock copolymers was performed on a Cary 400 Bio UV-Vis spectrophotometer (Varian, Palo Alto, CA), using a 0.1 cm path length quartz cuvette. Spectra were recorded in the scan mode, between 700 and 390 nm, with a 1 nm sampling interval. A 150 μM stock solution of CR was prepared in phosphate buffer saline (PBS; 50 mM Na2HPO4/NaH2PO4, 100 mM KCl, pH 7.0) and 10% ethanol to prevent CR micelle formation [30]. The acidic pH of P11 peptide solutions and the basic pH of peptide-polymer conjugates solutions in water (all 1 mg/mL) were adjusted to neutral pH by dilution with PBS (50%:50%, vol.-%). Consequently, the secondary structure of the new solutions was verified by CD and confirmed as being β-sheet. CR stock solution was added to these solutions for a final concentration of 3 μM CR. The resulting mixtures were incubated for 30 min at room temperature before the spectral analysis. 2.3.5. Congo Red binding studies CR birefringence studies were performed on an Olympus IX70 microscope equipped with a digital camera, at a magnification of 40×. Drops (~5 μL) of the mixture of P11 peptide and CR dye, and of the mixtures of P11-poly(HPMA)2k conjugate and CR dye, and respectively, P11-poly(HPMA)5k and CR dye, in PBS/ethanol 10%, used 56 for CR binding investigation by UV-Vis spectrophotometry, were applied to glass slides and left to dry in air for 30 min. The samples were observed under bright field illumination and then between crossed polarizers. Samples were prepared on copper specimen grids coated with a carbon support film (CF200-Cu; Electron Microscopy Sciences, Fort Washington, PA). The solutions of P11 at pH 2, and P11-poly(HPMA)2k and P11-poly(HPMA)5k at pH 11, having a β- sheet secondary structure as confirmed by CD, were used for sample preparation. The grids were placed with the carbon-coated face on top of a 5 μL drop of aqueous peptide solution (0.1 mg/mL; sonicated for 15 min in advance), for 30 s. The same procedure was followed for the preparation of the conjugates samples. Negative staining of the samples was done by placing the grids with the wet side down on top of a 5 μL uranyl acetate solution 4% (wt./vol.) for 20 s. The specimens were allowed to dry overnight, and then examined using a Philips Tecnai transmission electron microscope at 100 kV accelerating voltage. Random fields were photographed with magnifications ranging from 30000× to 150000×. 2.3.6. Transmission electron microscopy AFM observations were carried out on a modified Explorer AFM (Topometrix Inc., Santa Clara, CA) operating in contact mode, in air, at room temperature. Height images were recorded with silicon cantilevers (Mikromasch, Wilsonville, OR) at a resolution of 512×512 pixels (2 μm×2 μm). One drop of P11 peptide at pH 2 or conjugate 2.3.7. Atomic force microscopy 57 solution at pH 11 (0.01 mg/mL), possessing β-sheet structure as determined by CD spectroscopy, was sonicated for 15 min and placed onto the center of a freshly cleaved mica disc (Pelco, Ted Pella, Redding, CA). The resulting films were allowed to dry overnight, and then, their morphology was investigated. DI water with pH 2 and pH 11 (adjusted with either HCl or NaOH) were used as controls. AFM images obtained were processed using the WSxM software. 2.4. Results and discussion P11 β-sheet peptide, designed by Aggeli et al. [22], is a sequence rich in glutamine (Gln) residues. The distribution of glutamine, phenylalanine and tryptophan residues in P11 was designed to provide a hydrophilic face and a hydrophobic face that promote intermolecular recognition between adjacent P11 β-strands. Our interest in this well-characterized peptide was due to the fact that in acidic aqueous solutions (pH 2), it forms antiparallel β-sheets, which with increasing concentration, self-assemble into tapes, ribbons and fibrils [22, 31]. Secondly, P11 has a short amino acids sequence, which makes it easier to be synthesized by solid-phase method using manual Fmoc/tBu strategy. Indeed, a 96% yield of the P11 β-sheet peptide synthesis reaction was obtained, a good result, especially when considering the drawbacks of the process, associated with the aggregation of the β-sheet peptides on the resin. Part of P11 was acetylated, cleaved from the resin, purified and used for comparative studies with the P11-poly(HPMA) conjugates. The rest of the peptide-bound resin was modified at its N-terminus with maleimide group and used for the conjugation with the polymers. The poly(HPMA) 2.4.1. Synthesis of P11 peptide and P11-poly(HPMA) conjugates 58 polymers (poly(HPMA)2k, Mw=1990 g/mol, Mw/Mn=1.41, and poly(HPMA)5k, Mw=4890 g/mol, Mw/Mn=1.14) with thiol end groups were attached to P11 peptide via a thioether bond, according to the scheme illustrated in Figure 2.2. The copolymers were purified by dialysis and their purity was verified by HPLC (no free peptide was detected). Comparison of MALDI-TOF MS spectra of poly(HPMA) precursor and P11- poly(HPMA) conjugates, as well as their HPLC and SEC results, proved that the copolymers were obtained, although the yield of the coupling reactions was low (8% for P11-poly(HPMA)2k, and 6.66% for P11-poly(HPMA)5k, with respect to the pure conjugates), presumably due to a low accessibility of the thiol reactive end-groups as a result of their location inside the polymer chains [12, 16]. The peptide blocks in the copolymers were expected to form β-sheets under suitable conditions, and therefore, to impose their organization on the overall structure of the conjugates. Our hypothesis was verified by the results of the characterization studies presented below. 2.4.2. Circular dichroism spectra of P11 peptide and The secondary structures of P11 peptide and P11-poly(HPMA) conjugates were evaluated by CD spectroscopy. P11 peptide (1 mg/mL) in water at pH 2, incubated 3 days at room temperature had a β-sheet secondary structure, as described in literature [31, 32]. The intense negative maximum near 218 nm on P11 CD spectrum (Figure 2.3, A) is characteristic for peptides adopting a β-sheet conformation. P11-poly(HPMA) conjugates 59 Figure 2.2: Synthesis of β-sheet P11 peptide-poly(HPMA) diblock copolymers via thiol-maleimide coupling reaction 60 As suggested by Aggeli et al. [31], the slow kinetics of the antiparallel β-sheet formation was speeded up from days to only hours by adding seeds of pre-formed β-sheets to a newly prepared P11 solution at acidic pH (data not shown). However, CD spectra of aqueous solutions of P11-poly(HPMA)2k and P11- poly(HPMA)5k conjugates (2 mg/mL and 4 mg/mL respectively; the concentration of the peptide in the conjugate being thus kept approximately 1 mg/mL, 0.6 mM) at pH 2, after incubation at room temperature for 3 days, indicated a random coil structure (Figure 2.3, A). Even after incubation of the conjugates for one month, their secondary structure at pH 2 was unchanged. The same result was obtained at pH 7 (Figure 2.3, B). On the contrary, aqueous solutions of P11-poly(HPMA)2k and P11-poly(HPMA)5k at pH 11 (adjusted with NaOH) showed β-sheet secondary structures after being incubated for 10 days at room temperature, whereas P11 peptide in water at pH 11, after 10 days, had only a random coil structure (Figure 2.3, C). It seems that the concentration of the copolymers, the incubation time, the pH, and the presence of Na+ were essential for the formation of β-sheet structure. This observation was sustained by a series of simple experiments. As in the case of peptide solutions, the self-assembly could be speeded up; when the concentration of the copolymer was doubled, a β-sheet structure was obtained in water at pH 11, after only 8 days of incubation at room temperature. Interestingly, when the pH of an aqueous conjugate solution was adjusted to pH 11 using NH4OH instead of NaOH, in two attempts, the CD investigations evidenced only a random coil structure. This result is in agreement with data published by Zhang et al., according to which Na+ plays an important role in the self-assembly, whereas NH4 + does not induce the formation of β- sheets [33]. 61 Figure 2.3: CD spectra of P11 (filled circles), P11-poly(HPMA)2k (filled squares) and P11-poly(HPMA)5k (filled triangles) conjugates A. in water at pH 2, after 3 days of incubation at room temperature; B. in water at pH 7, after 18 days of incubation at room temperature; C. in water at pH 11, after 10 days of incubation at room temperature A B C 62 However, control experiments that were done on aqueous solutions of P11 and of its conjugates with HPMA in the presence of NaCl showed that Na+ alone, without the pH factor, is not able to induce the self-assembly. In order to explain the β-sheet formation in the P11-poly(HPMA) conjugates in aqueous samples at pH 11 adjusted with NaOH, the behavior of ionizable groups on the peptide, as well as the influence of polymer conformation, should be analyzed. At pH 11, Glu amino acid free in solution is negatively charged (pKa 4.1), whereas free Arg amino acid is positively charged (pKa 12.5); however, the pKa values of these amino acids in the β-sheet self-assemblies may be different due to the interactions with neighboring charged side chains [31]. Moreover, when, for comparison purpose, aqueous solutions of 2 mg/mL P11-PEG2k at pH 2 and pH 11, prepared following the same procedure as for P11-poly(HPMA) conjugates, were investigated by CD, β-sheets were detected at both pH values. However, for the same concentration of solutions, the intensity of the negative maximum near 218 nm largely increased as pH varied from 2 to 11, suggesting that in a conjugate, more P11 β-sheets are obtained in basic conditions. Therefore, given the random coil conformation of poly(HPMA) blocks versus the extended conformation of PEG blocks in the copolymers in water, P11 peptide was able to impose its β-sheet structure on the structure of the P11- poly(HPMA) copolymers only when strong electrostatic interactions were present. Favorable electrostatic interactions between positive side chains of Arg and negative side chains of Glu, conformation of poly(HPMA) block, as well as an active involvement of Na+, by binding to the γ-carboxylate of Glu, are presumably main factors responsible for the stabilization of the diblock copolymer β-sheets at pH 11. 63 Possible structural changes of the poly(HPMA) block at pH 11 in the low molecular weight copolymers P11-poly(HPMA)2k and P11-poly(HPMA)5k were also considered. Semitelechelic poly(HPMA) and P11-poly(HPMA) solutions (0.5 mg/mL) were prepared in water at pH 11 and incubated 10 days at room temperature. Comparison of HPLC spectra and SEC chromatograms of these samples before and after incubation showed no difference in the position of the peaks. Apparently, hydrolysis of HPMA side-chains, if any, was minor. Even though the hydrolysis of the maleimide to maleamic acid would occur in the conjugates samples at pH 11, this modification would not destroy the peptide-polymer linkage, but, instead, it would transform it into a thiol-maleamic acid bond. CR is a sulfonated azo dye that preferentially binds to proteins and peptides that are rich in β-sheets, similar to amyloids found in prion diseases. Given that the color of the CR changes from blue to red at pH 3-5.2, CR binding experiments were conducted at pH 7 in PBS buffer, only after CD investigations showed that the β-sheet structure was preserved in the new solutions after the pH change. CR binding indicated that peptide and the conjugates, P11-poly(HPMA)2k and P11-poly(HPMA)5k, bound to the dye, confirming the existence of β-sheet structure in these samples. CR binding to the β-sheets of P11 peptide resulted in a shift of the CR characteristic intense peak from 489 nm to 492 nm, whereas binding of CR to the conjugates produced a larger shift: to 500 nm for P11-poly(HPMA)2k, and to 508 nm for P11-poly(HPMA)5k (Figure 2.4, A). The point of maximal spectral difference (λmax) was estimated using the differential spectrum 2.4.3. Congo Red binding study 64 obtained by subtracting the absorption spectrum of CR from the absorption spectrum of P11 peptide/CR mixture, of P11-poly(HPMA)2k/CR mixture, and respectively, of P11- poly(HPMA)5k/CR mixture (Figure 2.4, B). A maximum spectral difference at 524 nm was obtained for the peptide, whereas 540 nm was calculated for both diblock copolymers. These values are a clear indication of the CR binding to the β-sheets in both cases, peptide and diblock copolymers, since λmax at 520-540 nm is a characteristic feature of β-sheets presence, as evidenced in β-amyloid fibrils [34, 35]. Green birefringence was detected under polarized light in peptide and both conjugates samples weakly stained by CR (samples from UV-Vis CR binding studies were used). In bright field, peptide/CR and conjugates/CR samples appeared yellow or red, depending on the amount of the CR dye. On the contrary, under crossed polarizers, same samples exhibited a green birefringence, an indication that the dye molecules bound to the β-sheets with a preferential orientation in respect to the β-sheet fibril axis (data not shown) [36, 37]. Visualization of the samples edges, where CR was not bound to the fibrils, showed no change in the red color under crossed-polarized light. 2.4.4. Morphology of the P11 peptide and P11-poly(HPMA) The self-assembly of P11 peptide and low molecular weight P11-poly(HPMA)2k and P11-poly(HPMA)5k conjugates was studied first by TEM. Micrometer-long fibrils were constantly observed by TEM imaging for P11 peptide (Figure 2.5, A), and P11- poly(HPMA)2k (Figure 2.5, B), and P11-poly(HPMA)5k (Figure 2.5, C) dried samples from aqueous solutions. conjugates self-assemblies 65 A B Figure 2.4: A. CR binding assay: absorbance spectra of CR alone (stars) and bound to β- sheets of P11 peptide (filled circles), P11-poly(HPMA)2k (filled squares) and P11- poly(HPMA)5k (filled triangles) conjugates; B. Differential spectra of ((P11/CR) - (CR)), (P11-poly(HPMA)2k/CR) - (CR)) and (P11-poly(HPMA)5k/CR) - (CR)) showing the points of maximum absorption 66 A B C Figure 2.5: TEM images of negatively stained A. P11 peptide sample in water at pH 2; B. P11-poly(HPMA