Recombinant DNA technology and click chemistry: a powerful combination for generating a hybrid elastin-like-statherin hydrogel to control calcium phosphate mineralization Mohamed Hamed Misbah1, Mercedes Santos1, Luis Quintanilla1, Christina Günter2, Matilde Alonso1, Andreas Taubert3 and José Carlos Rodríguez-Cabello*1 Full Research Paper Open Access Address: Beilstein J. Nanotechnol. 2017, 8, 772–783. 1G.I.R. Bioforge, University of Valladolid, CIBER-BBN, Paseo de doi:10.3762/bjnano.8.80 Belén 19, 47011 Valladolid, Spain, 2Institute of Earth and Environmental Sciences, University of Potsdam, D-14476 Potsdam, Received: 27 November 2016 Germany and 3Institute of Chemistry, University of Potsdam, D-14476 Accepted: 07 March 2017 Potsdam, Germany Published: 04 April 2017 Email: This article is part of the Thematic Series "Hybrid nanomaterials: from the José Carlos Rodríguez-Cabello* - [email protected] laboratory to the market". * Corresponding author Associate Editor: M. Stenzel Keywords: © 2017 Misbah et al.; licensee Beilstein-Institut. calcium phosphate; elastin-like recombinamers; hydroxyapatite; License and terms: see end of document. mineralization; SNA15 Abstract Understanding the mechanisms responsible for generating different phases and morphologies of calcium phosphate by elastin-like recombinamers is supreme for bioengineering of advanced multifunctional materials. The generation of such multifunctional hybrid materials depends on the properties of their counterparts and the way in which they are assembled. The success of this assembly depends on the different approaches used, such as recombinant DNA technology and click chemistry. In the present work, an elastin-like recombinamer bearing lysine amino acids distributed along the recombinamer chain has been cross-linked via Huisgen [2 + 3] cycloaddition. The recombinamer contains the SNA15 peptide domains inspired by salivary statherin, a peptide epitope known to specifically bind to and nucleate calcium phosphate. The benefit of using click chemistry is that the hybrid elastin-like- statherin recombinamers cross-link without losing their fibrillar structure. Mineralization of the resulting hybrid elastin-like- statherin recombinamer hydrogels with calcium phosphate is described. Thus, two different hydroxyapatite morphologies (cauli- flower- and plate-like) have been formed. Overall, this study shows that crosslinking elastin-like recombinamers leads to interest- ing matrix materials for the generation of calcium phosphate composites with potential applications as biomaterials. Introduction Combination of the specific properties of two materials is often tional performance that can be used for different applications, a key to generating a new material, whose properties are superi- such as tissue engineering [1]. This perspective can be applied or to those of its individual components, with improved func- in one of the hottest current research fields, namely control of 772 Beilstein J. Nanotechnol. 2017, 8, 772–783. the formation of calcium phosphate (CP) nanostructures for the bone tissue [20]. Although extensive research has been con- generation of biomimetic hybrid materials. Among these CP ducted on the mineralization field, it is a challenge to integrate structures, dicalcium phosphate dihydrate (DCPD), hydroxy- CP nanocrystals, different in morphology and composition, into apatite (HA) and β-tricalcium phosphate (β-TCP) have at- the hydrogel matrices [21]. tracted attention because of their potential applications [2,3]. HA is a stable crystalline phase that forms the main inorganic In order to obtain a hydrogel system that can control the forma- component of bone and teeth [2,4]. HA has a nanorod morphol- tion of CP, it is important to combine different approaches with ogy in natural bone, with the individual rods being roughly each other to overcome the challenges inherent to generating aligned parallel to one another throughout the collagen matrix materials with the desired properties. Recombinant DNA tech- [5-7]. β-TCP is a resorbable and degradable synthetic material nology [22,23] and click chemistry [24-26] are two such ap- that can be replaced by naturally re-grown bone tissue [2,3]. proaches. Thus, recombinant DNA technology offers advan- As a result, HA and β-TCP have already been used in tages such as reduction in production costs, a time reduction in (composite) materials for bone regeneration [2,8]. Due to the large-scale bioproduction, close control of the biomacromole- correlation between the (crystal) structure and properties of CP, cules product sequence (size and uniformity) and high yields it is important to be able to control its nanostructures [9-11]. For [22,23]. Moreover, it provides the possibility to achieve struc- example, hollow and mesoporous CP particles can be used for tural complexity by using various bio-inspired materials with drug delivery due to their high specific surface area and three- distinct mechanical, chemical or biological properties. Biomate- dimensional porous structures. In addition, osteoblast prolifera- rials that can be readily controlled using this approach include tion and apoptosis are affected by the size and shape of CP. the so-called elastin-like polymers (ELPs) or recombinamers Despite this interest, control of the formation of CP nanostruc- (ELRs), which are excellent example of materials that exhibit tures with specific characteristics remains a challenge. Al- self-assembly and self-organization [27-29]. though dry methods and high temperature methods are able to produce highly crystalline CP, they also produce aggregated The majority of ELPs or ELRs consist of simple amino-acid products of large crystal size and low phase purity [9,11]. More- consensus epitopes that are also present in natural elastin, such over, these processes cannot control the morphology and size of as (VPGXG)* (see Table 1 for details of sample nomenclature), the CP generated. Similarly, although wet-chemistry methods where the guest amino acid X can be any of the natural or syn- can be used to control the size and morphologies of CP under thetic L-amino acids except L-proline. ELRs exhibit an intrinsic mild reaction conditions, it is difficult to control the crys- inverse temperature transition (Tt) in aqueous solution. Thus, tallinity and phase purity of the synthesized CP nanostructures below Tt, the ELR chains are highly hydrated, which gives rise with narrow size distribution [9,12]. Furthermore, the genera- to well-solvated polymers in a random coil conformation, tion of delicate nanostructures, for example neuron-like mor- whereas above Tt, the ELR chains self-assemble into β-turns, phology of amorphous calcium phosphate (ACP) phase which are organized into a fibrillar morphology [27,30]. [9,12,13] remains a challenge due to the fast nucleation, Changes to the guest residue X modulate Tt, with the key influ- aggregation and subsequent anisotropic growth of the crystal ence being the polarity of the amino acid side-chain in X. As faces. such, ELRs can be designed with different polarities. This vari- ability provides access to a multitude of related, but different, Biomineralization-inspired “soft chemistry” routes provide amphiphilic multiblock ELRs with pre-programmed self-assem- simple and often rather cheap protocols for the synthesis of bling capabilities. Moreover, if the guest residue X carries complex CP-based hybrid materials with a high application amino or carboxyl groups, for example in lysine or glutamic potential [14]. Although organic additives can be used to acid, different post-synthesis reactions can be performed with control the CP mineralization process in aqueous solution, their the ELRs. soluble nature restricts their use in potential applications. To overcome this problem, organic chains are introduced as insol- As far as polymer bioconjugation is concerned, the thermore- uble additives, for example in the case of a templating ap- sponsivity of ELRs could be exploited to tune the bioactivity of proach or in a cross-linked state [15-17]. Following this ap- biological components [13,31-34]. According to this idea, the proach, the most common templates used are Langmuir mono- use of recombinant DNA technology [23,35] allows the combi- layers and self-assembled monolayers. Mineralization in gel nation of ELRs with mineralizing domains to be obtained. One phases has also attracted interest because these gels are easily of these mineralization-enhancing segments is the SNA15 prepared and provide a high level of control over mineraliza- (DDDEEKFLRRIGRFG)* peptide from salivary statherin that tion [18,19], including the potential to hierarchically generate has negative and positive charges stemmed from the side chains structured hybrid materials that may, for example, resemble of aspartic (D), glutamic (E), lysine (K) and arginine (R) amino 773 Beilstein J. Nanotechnol. 2017, 8, 772–783. acids [36]. The addition of specific functional domains further based on a hybrid elastin-like-SNA15 recombinamer. In this broadens the application potential of ELRs. The SNA15 domain regard, we are going to focus on the interplay between ELRs, exhibits a high affinity for CP and is able to nucleate mineral SNA15 and CP at the molecular level, in order to discuss the phases from calcium- and phosphate-containing solutions following outstanding inquiries: (1) The role of ELRs during [36,37]. Moreover, its α-helical conformation and distinct mineralization: How do they interact with the mineral? (2) The charge distribution