“Click” chemistry as a tool to create novel biomaterials: a short review

In recent years, there has been a growing demand for novel strategies for biomedical applications. Chitosan is a typical cationic amino-containing polysaccharide that has been widely used due to its unique properties. The grafting modification of chitosan has been explored as an interesting method to develop multifunctional novel chitosan hybrid materials for drug delivery, tissue engineering, and other biomedical applications. Recently, “click” chemistry has been introduced into the synthesis of polymeric materials with well-defined and complex chain architectures. The Huisgen’s 1,3-dipolar cycloaddition reaction between alkynes and azides yielding triazoles is the principal example of a “click” reaction. Bioconjugation, surface modification, and orthogonal functionalization of polymers were successfully performed through this chemoselective reaction. In recent literature interest has been shown in this cycloaddition for the modification of polysaccharides, however, only a few chitosan graft copolymers have been synthesized by this technique.


Introduction
In recent years, there has been a growing demand for novel strategies for biomedical applications. These strategies require biocompatible materials to serve as either structural support for tissue regeneration or delivery vehicles for cell and drug transplantation. This, however, is a challenging task due to the complexity of biological environment that impose the engineering of well-defined materials and surfaces (Nimmo andShoichet 2011, Azagarsamy andAnseth 2012). The recent progress in biotechnology and regenerative medicine has boosted the development of new synthetic organic techniques within regenerative biomaterials. In view of that, chemical conjugations are viewed as promising tools to not only to interrogate biomolecules in their native environment, but also to build materials for biomedical applications. However, most chemical reactions require long reaction times, strict exclusion of water, protection of the competing chemical groups, and vigorous heating/cooling or agitation. So, there has been a growing demand for engineering flexible, rapid, selective, and high yielding conjugation methods compatible with the complex biological milieu and physiological conditions (Nimmo and Shoichet 2011, Azagarsamy and Anseth 2012, Deng et al. 2011. These restricted and controlled reaction conditions were overcome with the advent of "click" chemistry. The term was introduced in 2001 by Sharpless et al. (Kolb et al. 2001) and chitosan was observed against a broad spectrum of microorganisms including fungi, algae and bacteria, being more active against Gram positive than Gram negative bacteria, in particular if it possess high molecular weight (Costa et al. 2012, Kong et al. 2010. Moreover, the reactive functional groups present in chitosan, namely the amino group at the C2 position of each deacetylated unit and hydroxyl groups at the C6 and C3 positions, can be readily subjected to chemical modification to generate a series of chitosan derivatives with new mechanical and solubility properties, enlarged biocompatibility and tailored properties (Goy et al. 2009, Leceta et al. 2013, Zhang et al. 2012. Interestingly, chitosan has the ability to be shaped in various forms such as films, microparticles, fibers and three-dimensional composite scaffolds for tissue engineering, all of them being largely used in different biomedical applications (Pandis et al. 2014, Ravi Kumar 2000, Rinaudo 2006. The properties of chitosan described above point out its great potential as a biofunctional material with promising results for tissue engineering and several other biomedical applications, such as drug and gene delivery (Croisier and Jérôme 2013, Carlson et al. 2008, Lieder et al. 2012, Costa et al. 2014, Shukla et al. 2013. The principal characteristics of chitosan and related effects are summarized and displayed in the next table (Table 1).

Main observed effects References Polycationic
Bioadhesive; interact with negatively charged GAG, heparin, proteoglycans, and polynucleotides like DNA or siRNA; antimicrobial activity; bacteriostatic effects and also analgesic effect Costa et al. 2012, Kong et al. 2010 Biocompatible Used in various medical applications, such as topical ocular application, implantation or injection

Bernkop-Schnürch and Dünnhaupt 2012 Biodegradable
Metabolized by certain human enzymes, especially lysozyme Berger et al. 2004b Promotes woundhealing Ability to foster adequate granulation tissue formation accompanied by angiogenesis and regular deposition of thin collagen fibers Costa et al. 2012, Kong et al. 2010 Amino and alcohol functions along chitosan chains Can be easily functionalized to further extend chitosan field of applications Goy et al. 2009, Leceta et al. 2013, Zhang et al. 2012 Table 1: Chitosan properties that make it an attractive polymer for biomedical applications 3. "Click" chemistry as a tool to create novel chitosan-based biomaterials 3.1 "Click" chemistry as a tethering tool "Click" chemistry reaction is a most promising, yet underexplored, approach which can be used for synthesis of various topological polymers or polymer modification (Iha et al. 2009, Wang et al. 2014. "Click" chemistry is a global term that refers to highly chemoselective and reactive reactions where two functional groups exclusively react with each other, even in the presence of other reactive functionalities. This bioorthogonal immobilization method has the ability to covalent assembly two chemically different molecules under mild conditions, such as complex carbohydrates with peptides, or chemoreporters, such as fluorescent dyes, with biopolymers. The reaction can be performed at room or physiological temperature and occurs in a manner that is high-yielding, rapid, modular, stereospecific, compatible with aqueous environments, and orthogonal to protecting and functional groups. So, the number of applications of "click" chemistry is growing in research areas, such as organic chemistry, bioconjugation, drug discovery, polymers, and radiochemistry (Nwe and Brechbiel 2009, Zeng et al. 2013, Amblard et al. 2009, Jiang et al. 2014, Nimmo and Shoichet 2011, Deng et al. 2011, Huerta-Angeles et al. 2012. Within the field of regenerative medicine "click" reactions have been used as bioconjugation techniques for decorating 2D cell substrates, and as promising patterning chemistries for immobilizing bioactive factors within 3D scaffolds (Nimmo and Shoichet 2011).

Huisgen's 1,3-dipolar cycloaddition
The Huisgen's 1,3-dipolar cycloaddition reaction between alkynes and azides yielding triazoles is the principal example of a "click" reaction and one of the most attractive chemoselective "click" reactions ( Figure 1). Azides and alkynes can be easily incorporated into a wide range of substituents and are stable under a variety of conditions. Both functionalities are also essentially inert to most biological and organic conditions, molecular oxygen, water, and the majority of common reaction conditions in organic synthesis. The potential of this reaction is very high, since alkyne and azide components react selectively with each other (Meldal and Tornøe 2008, Kolb and Sharpless 2003, Tron et al. 2008, Jagasia et al. 2009, Agalave et al. 2011). The unactivated azide-alkyne cycloaddition was first discovered by Huisgen in 1963 and required high temperatures and pressures, since azides and alkynes have low reactivity towards each other at atmospheric pressure and room temperature. This stability, being purely kinetic in origin, is responsible for the slow nature of the cycloaddition reaction and the inertness of these functional groups towards biological molecules and towards the reaction conditions inside living systems (i.e. aqueous, and mild reducing environments). Furthermore, as the directing effect of the substituents is usually weak, this reaction suffered from lack of regioselectivity, yielding a mixture of the 1,4-and the 1,5-triazole regioisomers, at times laborious to separate using classical chromatographic procedures. These issues were overcome by Tornøe and Meldal, who introduced Cu(I) catalysis in alkyne-azide coupling reactions, which led to a major improvement in both rate and regioselectivity; the copper catalyst favors formation of only the 1,4-regioisomer (Nwe and Brechbiel 2009, Meldal and Tornøe 2008, Kolb and Sharpless 2003, Tron et al. 2008, Amblard et al. 2009, Tornøe et al. 2002b, Rostovtsev et al. 2002, Deng et al. 2011). The formation of triazoles from azides and terminal alkynes catalyzed by Cu(I) is an extraordinarily robust reaction, which could be performed under a wide variety of conditions and with almost any source of solvated Cu(I). The most common conditions are the aqueous conditions employing copper sulfate (CuSO4) (Meldal and Tornøe 2008). The standard catalytic system uses copper (II) salts (e.g., copper sulfate pentahydrate or copper acetate) in the presence of a reducing agent, such as sodium ascorbate or metallic copper. This constantly reduces copper (II) to Cu(I) maintaining significantly high levels of the catalytic species (Tron et al. 2008, Castro et al. 2012). The triazole link created between the two building blocks (BB) that are coupled is very stable and chemically inert to most reactive conditions. In contrast to amides, and due to their high aromatic stabilization, triazoles cannot be cleaved hydrolytically or otherwise, and unlike benzenoids and related aromatic heterocycles, they are almost impossible to oxidize or reduce (Kolb and Sharpless 2003). These heterocycle have also an intermediate polarity with a high dipolar moment of ∼5 D and are able to participate actively in hydrogen bond formation as well as in dipole-dipole and stacking interactions (Meldal and Tornøe 2008, Agalave et al. 2011, Schulze and Schubert 2014, Chen et al. 2007). Finally, triazoles are also relatively resistant to metabolic degradation (Tron et al. 2008, Agalave et al. 2011) and, interestingly, have been found to display diverse biological activities, including anti-HIV, antituberculosis and anticancer activities and also antimicrobial activity against Gram positive bacteria (Tornøe et al. 2002a, Agalave et al. 2011).
As the archetypal example of "click" chemistry, the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition of azides and terminal alkynes (CuAAC) has been widely used in materials science and synthesis, molecular biology, and medicinal chemistry. Polysaccharides have been often modified using this reaction for different purposes such as creating new materials, new therapeutic or diagnostic agents, as a surface-modification strategy (Deng et al. 2011, Grischenko et al. 2015.

Chitosan-based biomaterials
Chitosan has been widely used as a drug delivery system, wound dressing, anticoagulant, and scaffold for tissue engineering owing to its properties. In recent years there has been a growing demand to broaden the applications of chitosan leading to the development of new techniques for enhancing its unique properties and develop multifunctional novel chitosan hybrid materials (Li et al. 2010, Bao et al. 2010b). However, applications of chitosan are limited by its poor solubility in water and in common solvents, being only soluble in acidic aqueous solutions where the amino groups are protonated. In this context, graft modification takes place as an interesting method to overcome this limitation. Moreover, this technique has also been explored in the development of multifunctional chitosan copolymers with well-defined structures and enhanced properties for specific applications (Kulbokaite et al. 2009, Zhang et al. 2012, Zampano et al. 2010, Munro et al. 2009). In this context, efficient and high yielding ''click" type reactions are useful and versatile tools.
In particular, the CuAAC to give 1,4-regioselectively substituted triazoles has attracted much attention for the modification of polymers. Indeed bioconjugation, surface modification, and orthogonal functionalization of polymers were successfully performed. In recent literature the interest has been shown in this cycloaddition for the modification of polysaccharides, however, only a few chitosan graft copolymers have been synthesized by this technique (Zampano et al. 2010). Stimuli-responsive drug delivery systems are been extensively investigated in order to improve drug delivery to a target site and therapeutic efficacy while minimizing side effects.
Biodegradable and biocompatible systems are been developed to respond to several stimuli, such as pH, temperature, redox or light (Guerry et al. 2014). Several polymers were already grafted to the chitosan backbone for the development of stimuli-responsive polymers. These polymers have attracted considerable interest for several biomedical applications such as smart bioactive surfaces, selective bioseparation, affinity control, or drug delivery. Poly(Nisopropylacrylamide) (PNIPAM) and poly[(2-dimethylamino)ethyl methacrylate] (PDMAEMA) were successfully grafted onto chitosan providing both temperature and pH responsive copolymer (Bao et al. 2010b, Bao et al. 2010a). Yuan and co-workers developed, via "click" chemistry, a novel amphiphilic chitosan graft copolymer that could assemble into spherical micelles with termosensitive properties . Micellar drug delivery systems have been widely investigated due to prolonged circulation in the blood, improved stability, and enhanced accumulation in tissues. Results showed that the chitosan graft copolymer micelles are promising candidates as intelligent carriers in controlled drug release system , Wu et al. 2009). According to the work of Zampano et al. selective functionalization and crosslinking of chitosan by azide-alkyne dipolar cycloaddition, showed to be particularly promising for the development of chitosan-based gels with pH-dependent swellability properties (Zampano et al. 2010, Uliniuc et al. 2012. A wide variety of biodegradable redox-sensitive amphiphilic copolymers has been described to incorporate and release drugs. In this context, amphiphilic chitosan-based graft copolymers containing a chitosan backbone and hydrophobic pendent blocks have been extensively studied (Guerry et al. 2014). "Click" reaction was used to synthesize amphiphilic graft copolymers composed of poly(ε-caprolactone) and chitosan. Their self-assembly in water, followed by core-cross-clicking with bis-alkynyl cystamine, led to redox-sensitive drug release nanoparticles. These drug-loaded cross-linked nanoparticles were effectively decross-linked in reductive environments mimicking cytoplasm and cell nucleus ones to release doxorubicin, an anticancer drug (Guerry et al. 2014). Bertoldo and colleagues reported the synthesis of a new light responsive polymer through CuAAC. Spiropyrans are a class of organic compounds with the ability to vary their chemical structure when exposed to light of an appropriate wavelength, and regain their original isomer when placed in the dark, or upon exposure to light of a different wavelength. The reaction between a 6-azido-6-deoxy, N-phthaloyl derivative of chitosan and a photochromic spiropyran resulted in a new chitosan based material with an interesting photochromic response to light irradiation (Bertoldo et al. 2011). A highly regioselective amphiprotic chitosan derivative, which can either donate or accept protons, was synthesized via "click" chemistry using 6-azido-6-deoxy chitosan derivative. The structure with positive (−NH3 + ) and negative (−COO − ) ions made the compound soluble under both basic and acidic conditions, respectively. Furthermore, it had the ability to form nanoparticles in neutral aqueous solution due to the inter-and intra-molecular aggregation. These properties make it possible to be used as drug delivery systems (Ifuku et al. 2013). CuACC was also applied to graft methoxy poly(ethylene glycol) (MPEG) onto N-azidated chitosan. PEGylation of polymers is one of the strategies used to overcome the uptake of polymer micelles by macrophage which is still a challenge for clinical applications since it lead to reduced half-time of the carrier in blood. PEG is a nontoxic and nonirritant hydrophilic polymer with the potential to sterically stabilize nanoparticles against protein absorption and subsequent phagocytosis thus improving the stability of the drug delivery system. This approach allowed obtaining chitosan copolymers with predetermined composition and simplified the purification process (Kulbokaite et al. 2009, Hu et al. 2008, Lin et al. 2011. In recent years, there has been a growing interest in the chemical modification of naturally occurring polysaccharides for developing non-viral gene vectors (Pang et al. 2015). Chitosan has been widely investigated for its cationic behavior and ability to bind to negatively charged molecules such as DNA, RNA, and oligonucleotide. Moreover, its low cytotoxicity has been demonstrated both in vivo studies and clinical trials. However, it is necessary to overcome the solubility limitation of chitosan and its low transfection efficiency. One of the routes to improve solubility is the regioselective modification of chitosan. Taking advantage of the concept of "click" chemistry, Gao and co-workers reported chitosan modification with a trimethylammonium cationic group through its hydroxyl groups in the C6 position. A non-toxic chitosan derivative was developed with increased DNA binding ability and cellular uptake suggesting that this new derivative could be an efficiently used for gene delivery (Gao et al. 2009, Zhao et al. 2013. CuAAC "click" reaction can also been used in the fabrication of layer-by-layer (LbL) ultrathin polymeric films and subsequent formation of microcapsules (De Geest et al. 2008). In the work of Zhang and colleagues this technique was used in the fabrication of chitosan microcapsules using LbL assembly. Moreover, this method allows postfunctionalization of chitosan with other materials, such as biomacromolecules, peptides or other functional groups thus creating microcapsules with additional properties, such as cellsspecific binding sites (Zhang et al. 2011). Tirino et al. synthesized a chitosan-based hydrogel by means of "click" chemistry with improved mechanical properties. Polyethyleneoxide (PEO) was selected owning to its biocompatibility, pH independent swelling and drug release characteristics. The synthesized hydrogel appeared to be suitable for several applications, such as drug release and wound healing due to its controllable crosslinking degree and ability to rapidly absorb high amounts of water (Tirino et al. 2014). Ryu and co-workers applied the CuCAA to the covalent functionalization graphene oxide (GO) sheets with chitosan. The functionalized graphene films displayed superior mechanical and highly enhanced antimicrobial properties make them competitive candidates for various biological applications (Ryu et al. 2013). Table 2 presents an overview of the different chitosan derivatives obtained through CuAAC "click" reaction.

Chitosan derivative Advantages
References Temperature and pH responsive copolymer composed of PNIPAM, PDMAEMA and chitosan Stimuli-responsive drug delivery systems Bao et al. 2010b, Bao et al. 2010a Amphiphilic graft copolymers composed of poly(ε-caprolactone) and chitosan Redox-sensitive drug nanoparticles to release an anticancer drug Guerry et al. 2014 Regioselective amphiprotic 6-azido-6deoxy chitosan derivative Nanoparticles to be used as drug delivery systems (Ifuku et   The CuACC reaction allows the design of various chitosan based materials with controlled and well-defined structures. Besides, this synthetic strategy enables further chemical modification at the residual positions of chitosan, which leads to the creation of high performance chitosan derivatives with the possibility of having several different functional groups at each position (Ifuku et al. 2012(Ifuku et al. , 2011.

Conclusions
Biological macromolecules, such as polymers, proteins and peptides, have been employed as indispensable biomaterials for decades. Polymers, in particular chitosan has been highlighted as a promising material due to its superior properties such as biocompatibility, chemical activity, abundance and relative low cost (Zhang and Cao 2014). The "click" chemistry concept has emerged as a unique and versatile family of highly efficient organic reactions for the synthesis of bio-inspired biomaterials that can be tailored according its final applications such as controlled drug release, gene delivery, tissue engineering and regenerative medicine. This chemical approach has become a powerful method within combinatorial chemistry due to its high efficiency, specificity, mild reaction conditions and tolerance to most functional groups. Within the "click" chemistry toolbox, CuAAC proved to be a highly versatile and useful reaction leading to the 1,2,3-triazole heterocycle (Loaiza et al. 2009, Nimmo and Shoichet 2011. As discussed in this review the CuACC reaction provides an attractive synthetic rout for welldefined biologically active chitosan-based moieties with specific properties. The synthesized polymers displayed different architectures namely block and graft polymers, films or hydrogels, tailored for each application. Over the past decade, this reaction has become a powerful approach within the biomedical and pharmaceutical fields to build materials that are cell compatible, highly functional and organized in structure (Azagarsamy and Anseth 2012, Chen et al. 2007, van Dijk et al. 2009). Bioorthogonal "click" chemistry has gained a significant impact in biomaterials design and synthesis over the last years, however, it is still an open field for researchers interested in biomaterials and their applications and it is expected that its importance will increase in the coming years (Azagarsamy and Anseth 2012, van Dijk et al. 2009, Nimmo andShoichet 2011).