Nucleoside-based supramolecular hydrogels: from synthesis and structural properties to biomedical and tissue engineering applications

1 Nucleoside-based supramolecular hydrogels: from 1 synthesis and structural properties to biomedical and 2 tissue engineering applications 3 Maria Godoy-Gallardo†,*, Maria Merino-Gómez†, Luisamaria C. Matiz†, Miguel A. Mateos-4 Timoneda†, F. Javier Gil†,§, Roman A. Perez†,* 5 †Bioengineering Institute of Technology (BIT), Department of Basic Science, International 6 University of Catalonia (UIC), Carrer de Josep Trueta, 08195, Sant Cugat del Vallès, Barcelona, 7 Spain 8 §Department of Dentistry, Faculty of Dentistry, International University of Catalonia (UIC), 9 Carrer de Josep Trueta, 08195, Sant Cugat del Vallès, Barcelona, Spain 10 KEYWORDS. Nucleoside-based hydrogels, guanosine and derivatives, supramolecular hydrogel, 11 self-assembly, stability improvement, enhancement of mechanical properties 12 ABSTRACT. Supramolecular hydrogels are of great interest in tissue scaffolding, diagnostics and 13 drug delivery due to their biocompatibility and stimuli-responsive properties. In particular, 14 nucleosides represent promising candidates as building blocks due to their manifold noncovalent 15 interactions and easiness of chemical modification. Significant progress in the field over recent 16 years has been made to allow the use of nucleoside-based supramolecular hydrogels in the 17 2 biomedical field, namely drug delivery and 3D bioprinting. For example, their long-term stability, 1 printability, functionality, and bio-activity have been greatly improved by employing more than 2 one gelator, incorporating different cations, including silver for antibacterial activity, or additives 3 such as boric acid or even biomolecules. This now permits their use as bioinks for 3D printing to 4 produce cell-laden scaffolds with specified geometries and pore sizes, as well as a homogenous 5 distribution of living cells and bioactive molecules. We have summarized the latest advances in 6 nucleoside-based supramolecular hydrogels, and we discuss their synthesis, structural properties, 7 and potential applications in tissue engineering, and provide an outlook and future perspective on 8 ongoing developments in the field. 9 1. INTRODUCTION 10 The development of advanced materials for tissue regeneration is a central goal in the field of 11 tissue engineering. Particular attention has been paid to the development of new biomaterial 12 supports capable to respond to both internal or external stimuli (e.g. pH, temperature, small 13 molecules, enzymes), and exhibit a controlled host immune response after implantation, among 14 others.1,2 Among the currently available biomaterials, hydrogels, i.e. three-dimensional (3D) 15 networks of hydrophilic and cross-linked polymers, are of particular interest owing to (i) their high 16 water content (typically 70–99% w/v) due to capillary effects and surface tension3; (ii) the 17 possibility to confer them with stimuli-responsive properties; and (iii), their 3D 18 microenvironments highly suitable for cell viability. 19 Depending on the underlying cross-linking mechanism, hydrogels can be defined as either 20 chemical or physical. Chemical hydrogels are created by covalent bond formation between the 21 individual polymer chains, and thus they provide a permanent matrix that is only disabled if the 22 3 bonds are broken. For example, cross-linking of vinyl-sulfone-functionalized dextran with 1 thiolated poly(ethylene glycol) (PEG) results in rapid formation of a chemical hydrogels.4 Physical 2 hydrogels, on the contrary, rely on non-covalent interactions such as hydrogen-bonds (H-bonds), 3 van der Waals forces, columbic interactions, and polymer chain entaglement.3,5 These interactions 4 reduce the flexibility of the system and drive gelation, thereby changing the macroscopic behavior 5 of the formulation, culminating in the formation of a 3D network. Two common hydrogels in the 6 biomedical field are polylactic acid (PLA) and polycaprolactone (PCL). However, as they are 7 rather hydrophobic, they are typically combined with hydrophilic polymers such as PEG to create 8 amphiphilic copolymers (e.g. PEG-PCL) with improved functionality.6 For further details on 9 frequently used non-nucleoside-based hydrogels, please refer to the following literature.7,8 10 Importantly, non-covalent forces are reversible and thus can be modulated by distinct stimuli 11 such pH change, or by the addition of bioactive molecules.3,5 Thus, reversibility of hydrogels may 12 be accomplished by reversible physical interactions (e.g. H-bonds, hydrophobic self-assembly and 13 host-guest interactions) or by dynamic covalent bonds (e.g. imine bonds, boronate ester bonds, 14 disulphide bonds, or via Diels-Alder reaction).9 15 The extracellular matrix (ECM) is an extensive and complex network of collagen, laminin, 16 elastin, and proteoglycans. Together with carbohydrates, they form a strong fibrous matrix via 17 intermolecular, non-covalent interactions that go beyond the individual molecule. Consequently, 18 a true biomimetic hydrogel has to exhibit similar supramolecular interactions as the ECM. In such 19 scenario, supramolecular hydrogels have to first form at the molecular level by self-assembly of 20 the components, followed by the establishment of a cross-linked network of non-covalent 21 interactions. Importantly, due to the high association and dissociation rates of the components, 22 these physical hydrogels exhibit strong dynamic behavior.10 According to their binding motifs, 23 4 supramolecular hydrogels can be primarily divided into (i) molecular hydrogels,11,12 (ii) polymeric 1 supramolecular hydrogels,10,13–15 and (iii) hybrid supramolecular hydrogels (Figure 2).16,17 2 Molecular hydrogels are materials formed by the hierarchical and non-covalent self-assembly of 3 low molecular weight gelators (LMWG, <3000 Da in molecular weight), including fatty acids,18 4 sugars,19 cholesterol,20 amino acids,12 peptides,21 and, most importantly, nucleosides.22 Such 5 hydrogels have attracted special interest due to their capacity to form fibrous networks mimicking 6 biopolymer networks by non-covalent association of their building blocks. Importantly, molecular 7 gelation occurs in three hierarchical steps: (i) the molecular building blocks converge to form one-8 dimensional nuclei; (ii) these nuclei then grow to form aggregates and nanofibers that can cross-9 link or entangle via non-covalent interactions; and (iii), the formed nanofibers then self-assemble 10 to form a complex 3D mesh and fibrillar network. Thus, the hydrophilicity and hydrophobicity of 11 the used gelators must be correctly balanced to permit and regulate the gelation of supramolecular 12 hydrogels. For example, too high hydrophobicity may lead to premature precipitation of the 13 hydrogel formulation and a too low capacity of the printed structure to hold water.23,24 Nucleic 14 acids such as nucleobases, nucleosides, and nucleotides represent attractive gelators and building 15 blocks for the synthesis of hydrogels due to their excellent biocompatibility and diverse 16 physicochemical properties, which can also be combined into binary hydrogels due to their shared 17 supramolecular chemistry and intermolecular interactions such as hydrogen bonds between 18 electron-deficient and electron-rich atoms.25 19 Polymeric supramolecular hydrogels are formed by polymers with supramolecular motifs, 20 resulting in their crosslinking. In the hybrid supramolecular hydrogels, nano-structural motifs are 21 used along with additional multivalent crosslinking sites. 22 5 These supramolecular hydrogels can remodel, reshape, and adapt to changing environmental 1 conditions.10 However, while their dynamic behavior allows them to be injected into target sites 2 or allows cells to spread and migrate, this dynamicity might decrease their mechanical strength. 3 Thus, the perfect balance between stability and dynamicity must be established for each potential 4 application. 5 The incorporation of orthogonal non-covalent interactions in their synthesis, such as π-π stacks 6 between aromatic rings, may provide a solution to this drawback.26–28 These additional attractive 7 interactions increase the strength and number of connections between the LMWGs, further 8 stabilizing the self-assembled hydrogel and greatly improving its mechanical properties. Among 9 the various hydrogel systems explored to date that exhibit these key characteristics, nucleoside-10 based hydrogels appear to be the most promising as they present a shear modulus and thixotropic 11 properties that are highly compatible with extrusion-based 3D printing, as they allow for the 12 embedding of both cells and bioactive molecules, and as they can ensure survival of extruded cells. 13 Furthermore, their chemistry is simple and non-hazardous, and their fibrillar and highly dynamics 14 network provides a soft scaffold that resembles the extracellular matrix, and their macroscopic 15 elastic behavior in combination with fluid-like microviscosity properties appears ideal for cell 16 proliferation and differentiation. 17 Recent studies have shown major developments in nucleoside-based hydrogels, including 18 enhanced therapeutic performance for drug delivery and controlled drug release. For example, 5′-19 deoxy-5′-iodoguanosine hydrogels were used to deliver antivirals,29 and mechanoresponsive 20 hydrogels based on thymine were employed for the administration of antibodies.30 To further 21 improve drug efficacy and reduce negative side effects on healthy cells or tissues, injectable 22 nucleoside-based hydrogels are being developed with sustained and tunable local release of 23 6 bioactive molecules. Notably, hydrogels have been used as so-called ECM-mimetics that can 1 provide a suitable environment for cell survival and proliferation due to their resemblance to soft 2 tissues and similarity in composition to the ECM.31,32 Furthermore, such hydrogels may be 3 prepared containing living cells and signaling molecules, further enhancing scaffold integration 4 and enabling capturing cells from circulation.33 Such supramolecular hydrogels have also been 5 shown to function effectively as scaffolds for bone tissue engineering. For instance, Maisani et 6 al.34 created hydrogels for cell embedding by combining glyco-nucleo-lipids containing a 7 fluorinated carbon chain (GNF) with type I collagen. After subcutaneous implantation, these 8 scaffolds provided a favorable environment for long term cell survival and promoted cell 9 differentiation into osteoblasts. 10 To summarize, ideal supramolecular hydrogels for tissue engineering can form under chemically 11 moderate or even physiological conditions, and they can repeatedly transition from primarily 12 elastic to predominantly viscous properties (shear-thinning), allowing them to regain their original 13 shape and viscosity upon stress.35 Such self-healing and re-assembly capabilities are critical for 14 allowing hydrogel injection at defect sites and to ensure minimal invasiveness during scaffold 15 implantation. Moreover, ideal hydrogels should enable encapsulation of both bioactive compounds 16 and living cells into their physically crosslinked nanofibrillar network, where a high-water capacity 17 and well-defined pores ensure cell survival, proliferation, and differentiation, while also allowing 18 for the extended and steady release of small molecule compounds such as drugs into the 19 environment, thereby augmenting such scaffolds (e.g.) with anti-inflammatory and/or anti-20 bacterial properties to improve implant effectiveness and minimize chances of rejection.36 Finally, 21 ideal hydrogels would enable tuneable drug release, for example by stimuli-responsive 22 capabilities, allowing their use as biosensors during tissue repair (Figure 1). 23 7 This review focuses on the most recent advances and developments in nucleoside-derived 1 supramolecular hydrogels, with a particular emphasis on the physicochemical properties important 2 in tissue engineering and regenerative medicine. With a focus on nucleoside-based hydrogels, we 3 discuss potential applications of different formulations as biomaterial inks, injectable hydrogels, 4 and cell-laden bioinks. In Table 1, a summary of the most important properties and applications of 5 nucleoside hydrogels is provided. We also address the need of developing and implementing 6 common quantitative metrics in the field to assure study comparability, as well as outline future 7 research directions and potential medical applications for nucleoside-based hydrogel materials. 8 2. NUCLEOSIDE-BASED SUPRAMOLECULAR HYDROGELS 9 Nucleosides are a class of biomolecules composed of a combination of a nucleobase 10 (nitrogenous base) and a sugar ring. Nucleobases are grouped into two major forms: purines and 11 pyrimidines (Figure 3Ai). While both are heterocyclic aromatic compounds, they diverge in their 12 chemical structure. Purines consist of a bicyclic structure with many potential H-bond interaction 13 sites, while pyrimidines have only one carbon nitrogen ring with three hydrogen donor and 14 acceptor sites.3 15 The ability of these building blocks to form supramolecular structures is imprinted on their 16 molecular level and their intrinsic properties such as proton affinities, hydrogen bonding and ππ 17 stacking capabilities, and the proper balance of hydrophobicity and hydrophilicity. Nitrogenous 18 bases, for example, can form supramolecular assemblies by forming H-bonds and π-π stack 19 interactions, which are critical for controlling the 3D structure of DNA and RNA assemblies.3,37 20 Moreover, they are frequently used as supramolecular building blocks to create more complex 21 structures with thixotropic (i.e., displaying a viscosity that lowers when stress is applied) and self-22 healing capabilities, all while maintaining a good environment for cell viability. Nucleobases have 23 8 gotten a lot of interest lately because of their exceptional ability to generate intermolecular 1 interactions in aqueous solutions and their use as building blocks of hydrogelators.38–40 2 Nucleobases readily engage in reversible interactions, and can form both homodimers and 3 heterodimers with other nucleobases. Notably, all five nucleobases, namely adenine (Ade), 4 cytosine (Cyt), guanine (Gua), thymine (Thy), and uracil (Ura) can form different enlarged 5 structures depending on the conditions, such as ribbons—a plane held together by eight hydrogen 6 bonds, or quadruplexes, a secondary structure with a central tetrad that can be further stabilized by 7 the presence of a cation like potassium (K+). The resulting dense network of physical interactions 8 not only infers hydrogel stability, but also structural versatility, tuneability, and self-healing 9 capacity, all key parameters of hydrogels for tissue engineering.41–43 10 Likewise, the ribose part of nucleosides can also play an important role in the formation of 11 supramolecular structures and thus contribute to their stabilization. For example, nucleotides 12 possess an anionic phosphate attached to the ribose ring that increases the strength of electrostatic 13 interactions (Figure 3Aii).44,45 Baldassarri and co-workers46 studied the formation of hydrogels by 14 guanosine 5’-monophosphate (5-GMP). The authors proposed that in the absence of external 15 cations, only the presence of 5-GMP monomer was detected, while the addition of cations (in 16 particular K+) provoked the self-assembly of the building blocks. Additionally, Sreenivasachary 17 and Lehn47 described the guanosine hydrazide as a powerful hydrogelator via the formation of 18 reversible acylhydrazone bonds after condensation with various aldehyde compounds, thus 19 creating dynamic libraries of acylhydrazones. As a result, the gelation process led to a multilevel 20 self-assembly of guanosine acylhydrazone derivatives into guanosine quartets (G-quartets). 21 2.1. Thymidine-based supramolecular hydrogels. While a number of nucleoside-based 22 hydrogels have been developed over recent years, only a few studies have focused on 23 9 supramolecular gels based on thymidine (Thd). For example, Yu et al.48 reported a binary hydrogel 1 based on Thd and melamine as a gelling component that formed a supramolecular complex gel via 2 H-bonds and π-π stacking interactions (Figure 4A). During the generation of the hydrogel, both 3 building blocks co-assemble into aggregates, and depending on the ratio of Thd to melamine, the 4 backbone forms shift from rod to sheet to curled slices, correlating with the amount of free -NH 5 groups. As a result, the authors were able to adjust the gelation properties and thus hydrogel 6 stability. These tunable changes in morphology allow the formation of nanotubular structures, 7 which might be exploited to make fibrillary self-assembled scaffolds. 8 Following this study, Ramin and colleagues49 used Thd as a basic building block to create a 9 stable supramolecular hydrogel by combining it with 1,2-dipalmitoyl-sn-glycerol phosphate 10 (diC16dT) and various monovalent ions such as lithium (Li+), sodium (Na+), K+, ammonium 11 (NH4+) and triethyl ammonium (Et3NH+) (Figure 4B). Experiments performed by transmission 12 electron microscopy (TEM) revealed that nanofibrillar structures were only found in diC16dT/Li+, 13 diC16dT/Na+ and diC16dT/K+, while lamellar structures were obtained in diC16dT/NH4+ and 14 diC16dT/Et3NH+. These results suggest that larger cations can impact the supramolecular 15 organization of the network. Moreover, these hydrogels possessed proper thixotropic and 16 rheological properties, allowing their use in injection-based applications such as 3D printing. 17 2.2. Cytidine-based supramolecular hydrogels. Cytidine (Cyd) nucleoside is another LMWGs 18 based on the Cyt nucleobase and a five-carbon sugar ribose with hydrogen-bonding donors and 19 acceptors which are necessary to induce gelation.50,51 However, despite the fact that Cyd has 20 intriguing characteristics like the capacity to bind to silver (Ag+), which could give the hydrogel 21 antibacterial properties, few studies have been reported on its potential as a gelator.52,53 This may 22 be explained by having one fewer hydrogen acceptor/donor group than guanosine (5 vs. 6), which 23 16 especially for tissue engineering.23,81,82 Importantly, because of the additional hydrogen-bonding 1 edges and its ability to create π–π stacks, guanine has the highest self-assembly capability of the 2 five nucleobases. 3 2.5.1. Guanosine derivatives hydrogels. To modulate the properties of G4 hydrogels, several 4 studies have suggested that the 5’-OH in the Guo is not important for the gelation, thus several 5 efforts have centered on 5’-modified guanosines, such as 5′-monophosphate,83–85 5’-hydrazides47 6 and 5’-OAc.86 Belda et al.87 studied the self-assembly of 5-GMP-derivatives using polyamines to 7 increase the stability of the 3D matrix. The results showed an increase of the gelation efficiency 8 when polyamines were used, and it was possible to reduce the 5-GMP concentration. Additionally, 9 Taylor et al.29 designed a self-destroying G4 hydrogel based on the self-assembly of borate esters 10 of 5-deoxy-5-iodoguanosine (5-IG). The authors hypothesized that the incorporation of the iodo 11 group (I) (a good leaving group) at C5’ of the ribose ring would facilitate the intramolecular 12 cyclization by N3. This may disrupt the integral structure of the gel since the newly formed bond 13 is incompatible with the G-quartet substructure. The results demonstrated that the in situ 14 intramolecular cyclization is temperature-sensitive and can occur even after gel formation, thereby 15 inducing “self-destruction”. Similar systems could be used as drug delivery carriers capable of 16 unloading their cargo in response to an external stimulus. 17 To address the low lifetime of Guo-based hydrogels, a series of studies focused on preparing 18 isoguanosine (isoGuo) hydrogels. IsoGuo is an isomer of Guo with an exchange of the substituents 19 at C2 and C6 (Figure 7A).88 Zhao et al.88 studied the hydrogel formation by using isoGuo combined 20 with low concentrations of alkali metal ions. Compared to previous Guo-based hydrogels, which 21 collapsed within minutes or hours, these derivative structures showed long-term stability for up to 22 several months. This was explained by the fact that isoGuo provides additional donor and acceptor 23 17 groups for the outer shell hydrogen-bond interactions. The authors concluded that the formed 1 tetraplexes and pentaplexes were piled up to form aggregates, while the outer shell H-bonds 2 provoked the ribbon-like interaction with other isoGuo compounds. Scanning electron microscopy 3 (SEM) demonstrated that isoGuo hydrogels formed interconnected helical fibers and that, under 4 physiological conditions, the gel showed long-term stability and exhibited great loading and 5 release properties for small-molecule compounds. In particular, the authors encapsulated a cationic 6 and aromatic dye, namely methylene blue (MB), in the isoGuo hydrogel, showing a relatively fast 7 release of ~40% in the first 4 h. Then, the remaining MB was released slowly, leading to 8 equilibrium after 26 h. Therefore, the use of the isoGuo biological activity together with its gel-9 forming properties may be especially useful in the context of drug delivery applications or for the 10 construction of nanoscopic devices. 11 Thakur et al.89 developed a dual cross-linked G4 hydrogel capable of controlling the delivery of 12 doxorubicin (Dox), a potent anticancer drug. In particular, the authors formed a G4 hydrogel by 13 cross-linking the G4 of the nucleotide 5-GMP through interactions between the phosphate groups 14 and hydrolyzed iron (III) ions (Fe3+). Finally, the hydrogels were cross-linked through calcium ion 15 (Ca2+) by bridging the diol groups of the ribose moiety of 5-GMP. In an in vitro study of 20 days, 16 the resulting hydrogel showed a pH-responsive release of doxorubicin. For that, the authors 17 immersed the hydrogels in phosphate buffered saline (PBS) (10 mM, pH 4.0, 5.5, and 7.4) and at 18 different time points, the PBS buffer was removed for analysis, and replaced with fresh buffer. 19 After 20 days, only 3% of Dox was released at pH 7.4, suggesting high stability of the gel. 20 However, at pH 4, a much faster release was observed (90% at day 20), demonstrating the acid-21 responsive behavior of the hydrogel. Due to this responsiveness to an external stimulus and its 22 constant rate of drug release, these hydrogels might enhance the versatility of drug delivery 23 18 systems. An important limitation of Guo-based hydrogels is due to the potential crystallization of 1 the gel structure, which can happen within hours at room temperature and strongly impacts their 2 stability, as the gels are rendered brittle and lose their flexible overall structure. Feng et al.90 3 detailed the crystallization mechanism of a 2’-deoxy-2’-fluoroguanosine (FGd) hydrogel in order 4 to understand the balance between gelation and crystallization. The authors confirmed that the 5 addition of K+ into the G-quartet facilitated the formation of a linear ribbon structure, and they 6 suggested that it may be related to the tendency of Guo-based hydrogels to crystallize. 7 2.5.2. Binary hydrogels of guanosine with other derivatives. A number of binary hydrogels 8 constituted by Guo and one of its derivatives have been recently assessed in order to increase their 9 lifetime stability and physical properties.91,92 Among them, the mixture of the hydrophobic Guo 10 and the hydrophilic 5-GMP was able to form stable and transparent hydrogels in water. Guo is 11 characterized as an insoluble nucleoside that can form stable hydrogels even at high salt 12 concentrations. On the other hand, 5-GMP is highly soluble at neutral pH but its gelation is highly 13 concentration dependent. Hence, 5-GMP improves the solubility of Guo, and the insolubility of 14 Guo endorses the gelation of 5-GMP at lower concentrations. Carducci et al.93, for example, 15 described a binary mixture of Guo and 5-GMP capable of forming a transparent hydrogel in the 16 presence of K+. Next, Navas et al.82 combined Guo and 5-GMP in order to obtain a two-compound 17 hydrogel and to study the mechanism causing the gelation of the solution. The authors described 18 a nano-partitioning of the mixture where the 5-GMP-rich region forms large columns of G-quartets 19 while the Guo-rich areas showed knotted coils of G4. The Guo-rich tangles formed the nodes of 20 the network and were linked through the thermally reversible 5-GMP-rich fibres. Later, Yoneda 21 et al.94 studied the encapsulation and release of key proteins from the hydrogel using MB as a 22 photosensitizer (Figure 7Bi). They employed a combination of Guo and 5-GMP and loaded the 23 19 hydrogel with MB at a final concentration of 50 mg mL-1. Their analysis revealed a hydrogel with 1 good mechanical properties that had no cytotoxic effects on cells when seeded onto the matrix. 2 Moreover, the MB release study suggested a mechanism dependent on the hydrogel composition. 3 For example, the release of MB after the first 8 h was highest when the ratio or Guo-to-5-GMP 4 was 1:6 (57%), followed by 1:2 (46%) and finally 1:1 (31%) (Figure 7Bii). However, after 24 h, 5 the MB release from the 1:6 ratio was the lowest. This was explained by the fact that these 6 hydrogels had the highest amount of negative charge in the hydrogel (due to the highest 7 concentration of 5-GMP), and this may have sequestered some basic levels of MB molecules due 8 to their positive charge. And importantly, it has been demonstrated that binary hydrogels of Guo-9 to-5-GMP may be employed as biomaterials for biomolecule encapsulation. 10 While isoGuo is capable to form stable hydrogels, it exhibits poor injectability due to insufficient 11 strength after injection, limiting its applicability in tissue engineering applications. Taking this into 12 account as well as the properties of Guo for generating G-quadruplex hydrogels, Tang et al.56 13 evaluated the utilization of Guo and isoGuo in the presence of cations to form a two-component 14 co-gel. The results showed that a self-healing nucleoside hydrogel containing K+ may be created. 15 SEM images revealed a flower-like morphology and evidenced the presence of some cross-linking 16 between Guo and isoGuo (Figure 7C). Injection experiments further demonstrated that the co-gel 17 had a quick recovery time and overall injectable properties. Finally, isoGuo is considered as a 18 molecule with antitumor activity.95 As a result, this two-component co-gel has the potential to be 19 effective not only in tissue regeneration but also in anticancer therapy. 20 2.5.3. Incorporation of different ionic species in guanosine hydrogels. To avoid detrimental 21 crystallization, Feng and colleagues90 proposed that stronger metal bonds (e.g. platinum ion (Pt2+), 22 mercury ion (Hg2+) or Ag+ could be a promising alternative due to the formation of a metal ion-23 20 linked and H-bonded structure by coordination with the electron-rich nitrogen and oxygen groups 1 from the surrounding heterocyclic purine rings. Hence, the FGd hydrogel was prepared in the 2 presence of silver ions and thereby demonstrated a tremendously increased lifetime stability of up 3 to 6 months (Figure 7Di and ii). Furthermore, the use of silver ions in the hydrogel formation may 4 provide antibacterial properties to the network, making applications in wound healing a logical fit 5 (Figure 7Diii). 6 Similarly, recent studies support the idea that lanthanides or trivalent ions can induce Guo 7 gelation.89,96 Zhang et al.97 used 5-GMP as building blocks to achieve gelation via G-quadruplex 8 formation in aqueous solution by the use of lanthanide ions, such as lanthanum (La3+), cerium 9 (Ce3+), europium (Eu3+) and terbium (Tb3+). The results showed an increase in the stability of the 10 G4 hydrogels when lanthanide ions were used instead of the traditionally used monovalent cations. 11 The TEM images showed a denser arrangement of nanofibers induced by La3+ and Ce3+ compared 12 to Eu3+ and Tb3+, which displayed short nanofibers or crowded clusters. These distinct 13 microstructures were correlated with the mechanical strength of the hydrogel, reaching its highest 14 when La3+ was used at a concentration of 12 mM. The authors associated this effect with the 15 interaction degree of the La3+ with the G-quartets via ion-dipole interaction, which affects the 16 gelation properties of the gels. 17 To date, a number of studies have been performed to examine Guo-based hydrogels formed with 18 divalent cations (M2+) instead of traditional alkali metals.23,98,99 Although the majority of these 19 studies focused on hydrogels for environmental purposes, Plank and colleagues80 hypothesized 20 that the greater charge density given by M2+ would improve the electrostatic interactions with the 21 anionic gelators. In particular, they studied the formation of stable and transparent Guo hydrogels 22 21 using different divalent cations such as barium (Ba2+), strontium (Sr2+) and lead (Pb2+) by mixing 1 Guo with 8-aminoguanosine (8AmG). 2 2.5.4. Guanosine hydrogels with boric acid and its derivatives. In recent years, boronic acids 3 (BAs) have become a powerful addition to improve the stability of G4 supramolecular hydrogels. 4 Peters et al.39 described a Guo-based hydrogel where the right combination of borate anions and 5 K+ was crucial for gelation and stability. After combining Guo, potassium hydroxide and boric 6 acid, a guanosine-borate (GB) hydrogel with excellent lifetime properties was obtained. The 7 authors hypothesized that a single borate anion is able to react with Guo in order to form covalent 8 Guo dimers that act as building blocks in the supramolecular network where the GB diesters 9 facilitate the self-assembly of G4-K+ thus the formation of stable hydrogels. The same research 10 group subsequently provided further insights into the mechanism of GB hydrogel formation by 11 studying how the individual components (e.g. borate salt, GB diesters or G-quartets) determine the 12 structure and properties of the final GB hydrogels.55 For example, the authors observed that the 13 addition of K+ gave the strongest GB hydrogel in comparison to Li+. By the fluorescence response 14 of thioflavin-T (ThT), a dye that selectively binds to the G-quartet, the authors observed the largest 15 fluorescent signal when K+ was used for gel formation (Figure 8A). The authors hypothesized that 16 this was due to increased and more robust G-quartet generation in the presence of K+, as evidenced 17 by the increased number of ThT binding sites. The results showed that the cations' importance 18 relies on the stability of the anionic diesters and the G-quartet units, that are the real building blocks 19 of the supramolecular hydrogel. The results showed that the type of cation strongly impacts the 20 stability of the GB diester and G-quartet and, thus, as the latter represent the crucial building block 21 of the gel, the properties of the supramolecular hydrogel. 22 22 Inspired by the use of boronate compounds for hydrogel stability, Qiao et al.100 described the 1 generation of a GB-based hydrogel by the combination of Guo and 2-formylboronic acid (2-FPBA) 2 in the presence of potassium hydroxide (KOH). Intriguingly, the authors reported difficulties in 3 forming a self-supporting gel when alkali metal ions were used instead of K+. In 2020, Ghosh and 4 co-workers101 reported the use of 1-naphthaleneboronic acid (NapBA) to form a Guo-based 5 hydrogel. As shown by fluorescence and circular dichroism (CD), the generation of G4 structures 6 was discriminating towards K+ ions (Figure 8B) Moreover, after the evaluation of the 7 biocompatibility of the hydrogel by in vitro cytotoxicity, the authors studied the quantitative and 8 controlled release of vitamin B2 and vitamin B12. The results showed a higher release rate for 9 vitamin B2 than vitamin B12 (60% and 30% for the first 40 h, respectively), probably due to the 10 nearly 4-times larger molecular weight of vitamin B12. Thus, the authors could clearly demonstrate 11 that these hydrogels are not only useful for tissue engineering but also for controlled drug delivery. 12 The multiple and dynamic interactions in the supramolecular network allow the hydrogel to 13 respond to various stimuli such as heat, pH, glucose, and hydrogen peroxide. Li et al.79, for 14 example, reported a stimuli-sensitive GB hydrogel composed of Guo, 2-FPBA, tris(2-15 aminoethyl)amine (TAEA) and a K+ core. The 2-FPBA was added to the network both to enhance 16 hydrogel formation and to provide a controlled breaking point upon inducible and dynamic 17 interactions under pH changes and saccharide structure interactions. Additionally, Hu et al. 77 18 detailed a multi-stimuli responsive hydrogel consisting of Guo, K+, 2-FPBA and aminoglycoside. 19 Notably, aminoglycosides present potent broad-spectrum antibiotic action. In this study, the 20 authors found that they also act as good hydrogel gelators, potentially facilitating antibacterial 21 wound patches for the treatment of bacterial infections.In 2020, Biswas et al.102 reported the 22 preparation of a dynamic GB hydrogel by combining Guo, 2-FPBA and 4-Arm PEG-NH2, a 23 23 multiarm PEG derivative with terminal amine groups and a pentaerythritol core. The authors 1 hypothesized that the imine bonds acted as a connector between the PEG and the G-quartet units, 2 resulting in a highly intertwined dynamic network and a G4 structure, which was confirmed by 3 ThT binding. When ThT was added to the GB hydrogel, it became fluorescent and showed a 4 distinct emission peak at 560 nm (λex = 450 nm), which was caused by the π-π stacking interactions 5 between ThT and the G4 structure. Additionally, a self-healing test was conducted by connecting 6 three pieces where only the external ones were treated with ThT. The results showed a gradual 7 diffusion through the connecting joints, and after 2 h, a fused hydrogel block was observed. 8 Finally, the authors incorporated the anticancer drug doxorubicin hydrochloride and monitored the 9 time-dependent drug release. Upon shifting the pH to 4.8, the acid-labile imino-boronate bonds 10 were broken, leading to the destabilization of the hydrogel nanofibers and, consequently, the 11 release of the embedded drug. This study elegantly demonstrated the use of these stimuli-12 responsive hydrogels as a promising strategy for cancer therapy. 13 Different approaches have been explored to construct multicomponent GB hydrogels by adding 14 additional small molecules, especially divalent ions. Rotaru et al.74 prepared G-quartet hydrogels 15 by combining Guo with benzene-1,4-diboronic acid (BDBA) using both K+ and Ba2+ ions, and 16 then further aiding cross-linking with magnesium (Mg2+) ions. The resulting hydrogel showed 17 increased stability and was able to sustain higher water retention, which improved the cell growth 18 properties. Cell growth and viability tests were performed using normal human dermal fibroblasts 19 (NHDF) and seeding them onto the hydrogel. Cells showed a viability of up to 73% after 24 h. 20 Wu and colleagues22 studied the incorporation of the antiviral drug acyclovir (Acv) and the 21 loading of the anti-cancer drug methotrexate (MTX) in a GB hydrogel using Na+ for G-quartet 22 formation, and they were able to obtain homogeneous MTX/GB and Acv/GB hydrogels, 23 24 respectively. 1H-NMR analysis revealed that Acv had been inserted into the G4-quartet structure 1 and had replaced Guo molecules. Moreover, CD measurements demonstrated that Acv reduced 2 the interaction strength between the boric acid ester and the Guo fibers, leading to a weakened 3 hydrogel with lower mechanical strength. Due to the responsive properties of GB hydrogels to 4 e.g., glucose and pH, the induced release of Acv and MTX was monitored. MTX exhibited an 5 initial burst followed by a slow release. Due to the lack of interaction between the MTX and the 6 hydrogel, the strength of release solely depended on the intensity of the external stimulus (pH, 7 glucose and Na+ concentration). However, as Acv was part of the G4-quartet, the initial release 8 was much lower compared to MTX, and under all conditions tested, the total release of MTX was 9 higher than Acv. Therefore, by adding moieties to drugs that are able to interact with the G-10 quartets, the release rate of the drug may be altered and controlled, allowing the encapsulation of 11 distinct biomolecules at the same time but with different release profiles. 12 2.5.5. Incorporation of compounds in guanosine hydrogels. The development of hydrogels for 13 faster tissue regeneration is of the utmost importance, and one key approach is to incorporate 14 functional agents into the matrix. For example, Xiao et al.75 reported the incorporation of 15 recombinant human collagen (RHC) into G4 hydrogels for creating medical patches for wound 16 repair. The authors postulated that the flexible G4-RHC hydrogel does not only cover the wound, 17 but releases the entrapped RHC, thereby recruiting macrophages and fibroblasts, and thus 18 accelerating the epithelial regeneration (Figure 8Ci). In vivo studies showed that the flexible RHC-19 G4 hydrogel was more effective than agarose -based patches to stimulate wound healing. For 20 example, after 7 days of treatment, the wound area was about 91% sealed with the RCH-G4 patch 21 while only 86% wound closure was obtained for the agarose control group (Figure 8Cii). 22 25 Recently, Li et al.103 proposed and demonstrated that the cationic surfactant 1 cetyltrimethylammonium bromide (CTAB) could promote the formation of G4 gels. In particular, 2 CTAB interacts with nucleotides, such as 5-GMP, helping to form and modulate the transition of 3 G-quadruplex structures. This potentially groundbreaking finding opens up new possibilities for 4 fine-tuning functional materials that are based on G4 assemblies. 5 2.6. Heterotypic nucleoside-based supramolecular hydrogels. The majority of hydrogels are 6 generated by using a single gelator, but hydrogels composed of two or more supramolecular 7 building blocks may show certain benefits, such as a greater degree of adaptability.104 Wang and 8 co-workers105 studied the gelation of bis-quaternary ammonium gemini surfactant with 5’-uridine 9 monophosphate (5-UMP). The results showed that the gelation of the mixture did not occur at a 10 concentration of 10 mM for the complex, but the addition of an equimolar amount of Ado turned 11 the solution into a supramolecular hydrogel. The authors proposed that the hydrogelation was due 12 to the formation of H-bonds between 5-UMP and the Ado by hydrophobic interactions between 13 the nucleobases. Importantly, such a molecular recognition tool resulting in hydrogel formation 14 might show great promise and applicability for biosensors. 15 In general, hydrogels contain a high amount of water, which produces weak but significant 16 adhesion between the hydrogel and solid surfaces. Importantly, adhesive hydrogels have proven 17 broad applicability in tissue engineering; for example, they are used in electronic devices for the 18 human body,106 to repair damaged tissues,107 in the 3D printing of tissues108, and as bio-glue.109 19 Liu et al.110 presented a hydrogel composed of acrylated adenine and acrylated thymine. The 20 adhesion between the hydrogel and the solid substrate was improved by the presence of A and T, 21 and therefore, the generation of structures via H-bonds, metal-complexation, and hydrophobic 22 interaction between the nucleobases with the compounds of the solid. In particular, A and T were 23 32 showed a set of fibers with a thickness of between 50 and 100 µm (Figure 10C). For cell viability 1 studies, cells were incorporated into the hydrogel by mixing with a 39 °C preheated gel. The 2 printed cell-laden hydrogel had a low cytotoxic effect on human gingival fibroblasts (HGFs, 93.5% 3 viability on day 1) but a slightly higher cytotoxicity on stem cells from the apical papilla (SCAPs, 4 70.5%) (Figure 10D). The authors confirmed previous results that showed that HGFs and SCAPs 5 were resistant to shear stress and pressure during the extrusion process. The reduced SCAP 6 viability was attributed to the higher fragility of the stem cells, complicating their ability to survive, 7 especially in the center of the printed samples. Importantly, the in vivo study did not show any 8 evidence of inflammation, demonstrating the high in vivo tolerance of the printed scaffold. Based 9 on these results, we anticipate that this and other LMW hydrogels will be employed as customized 10 implants for soft tissue reconstructions, which, to the best of our knowledge, has not been 11 attempted so far. 12 Over the last years, important advances have been achieved, including the formulation of hybrid 13 multicomponent hydrogels, and cell-laden Guo-based hydrogels showing great potential as a 14 platform for tissue regeneration. However, strategies need to be developed to improve the cell 15 viability of the encapsulated cells and to guarantee their protection during the printing process. 16 This could be achieved by providing improved viscoelastic properties to the hydrogel, which 17 would allow the appropriate fluidity in the nozzle to ensure cell protection. Additionally, as the 18 field evolves, we expect that the number of applications entrapping additional biomolecules to, 19 e.g., stimulate vascular network formation in the 3D constructs will increase. 20 Unfortunately, many extrusion-based (bio)printing techniques exhibit insufficient printing 21 accuracy to allow a proper evaluation of the printability of the (bio)ink. Furthermore, many studies 22 either do not evaluate or merely test the printability of their new (bio)inks, or assess it only in a 23 33 poor qualitative manner. Importantly, first efforts have been made and preliminary measures have 1 been established in the field to determine, for example, shape fidelity, printing accuracy, and 2 extrudability of new materials in a more quantitative way. However, each of these techniques 3 evaluates the (bio)ink in a unique way, making comparison difficult, and many studies still rely on 4 methods with few or no measurable metrics. Similarly, descriptive terms such as “printability” 5 require a better and broadly accepted definition, and quality control procedures need to be 6 standardized to improve comparability. 7 Furthermore, computational prediction and simulation approaches have gained momentum in the 8 last decade since they can help us to comprehend the underlying self-assembly properties and thus 9 to enhance the performance of supramolecular hydrogels.129,130 Such computational techniques can 10 also provide information on time points and scales of the gelation process that experimental 11 methods cannot easily provide, allowing us to obtain a more complete picture of the process, 12 especially when multiple computational methods are combined and a variety of models with 13 varying levels of resolution and complexity (multiscale modelling) are used.131,132 In particular, 14 the use of machine learning 133–135 and AI-based prediction tools136,137 will significantly accelerate 15 the development of application-tailored supramolecular hydrogels since potential gelators can be 16 first thoroughly assessed in silico before advancing into experimental trials. Such in silico analysis 17 would ideally yield a comprehensive, freely accessible database comprising all calculated and 18 predicted self-assembly properties of the various gelators, which would be a tremendous resource 19 for the field.54,130 20 Without doubt, the development of robust evaluation standards in the field is of the utmost 21 importance to allow a comparison of different hydrogel compositions, printing techniques, and 22 cell entrapment strategies. This will aid in the development of new application-specific 23 34 formulations and will considerably advance the field by allowing a better understanding of the 1 underlying mechanics of bioink printability. 2 3 35 1 Figure 1. Properties and applications of ideal supramolecular hydrogels in the field of tissue 2 engineering and biomedicine. Reproduced with permission from ref 36. Copyright 2021 MDPI. 3 Reproduced with permission from http://smart.servier.com/. Copyright 2022 Servier Medical Art. 4 5 36 1 Figure 2. (A) Noncovalent interactions involved in the formation of supramolecular hydrogels. 2 (B) Classification of supramolecular hydrogels based on the type of building block: (i) molecular 3 hydrogel, (ii) supramolecular polymeric hydrogel and (iii) supramolecular hybrid hydrogel. 4 5 37 1 Figure 3. (A) (i) Molecular scaffold of nucleosides and nucleotides. Covalently, attachment of a 2 nucleobase to a D-ribose via a glycosidic bond forms a nucleoside. When one or more phosphate 3 groups are present, it refers to a nucleotide. The phosphate (green) group can be utilized for 4 electrostatic interactions in the hydrogel network. (ii) Molecular schematics of DNA and RNA‘s 5 five nitrogenous bases (nucleobases). (B) Hydrogen donor (orange arrow) and acceptor (blue 6 arrow) groups of guanosine provide distinct pathways for molecular recognition or self-assembly. 7 (C) Hoogsteen and Watson-Crick face of guanosine. Guanine and guanine derivatives may arrange 8 into (D) guanosine quartets (G-quartet) and (E) guanosine ribbons (G-ribbons) in the presence or 9 absence of metal ions, respectively. 10 11 38 1 Figure 4. (A) Proposed self-assembly mechanism of thymidine (Thd) and melamine (M) 2 hydrogels at different molecular ratios form rods, sheets, and flower-like structures (B) Proposed 3 supramolecular organization of helical fibers in nucleolipid-based hydrogels using thymidine-4 functionalized dipalmitoyl-sn-glycerol phosphate (diC16dT). Worm-like micelles are formed in 5 the presence of small counter ions (Li+, Na+, K+) via self-assembly. TEM images of (i) non-6 functionalized 1,2-dipalmitoyl-sn-glycerol-3-phosphate versus (ii) thymidine-functionalized 7 DiC16dT (6% w/w, 0.9% NaCl). Inset shows a higher magnification image. Panel A reproduced 8 with permission from ref 48. Copyright 2011 Elseiver. Panel B reproduced with permission from 9 ref 49. Copyright 2017 Wiley-VCH. 10 11 39 1 Figure 5. (A) (i) Proposed molecular structure of the cytidine borate diesters that self-assemble 2 through electrostatic interactions via silver ions (Ag+) to form supramolecular hydrogels. (ii) A 3 proposed polymerization mechanism for cytidine borate diesters that self-assemble to form 4 hydrogels. (iii) Top: atomic force microscopy analysis of a hydrogel made up of nanowires (scale 5 bar 200 nm). Bottom: a bridge is formed by extruding the hydrogel (5.6 wt% cytidine and dyed 6 with methylene blue) with a syringe under shear stress (scale bar 5 mm). (B) Antimicrobial 7 activities of silver-stabilized cytidine borate hydrogels against various bacteria. (i) Hydrogel-8 dependent inhibition of E. coli, P. aeruginosa and S. aureus on LB agar plates is shown. (ii) Dose-9 dependent reduction of E. coli, P. aeruginosa and S. aureus colony-forming units (CFUs). 10 Reproduced with permission from ref 51. Copyright 2019 American Chemical Society. 11 40 1 Figure 6. (A) Structural schematic showing the chemical coordination between AMP and Ag+. 2 (B) Photographic comparison with different concentrations of AMP and Ag+. (i) Constant AMP 3 concentration of 40 mmol L-1 with increasing Ag+ concentrations from left to right (mmol L-1): 5, 4 10, 20, 30, 40, 50, 60; (ii) Constant Ag+ concentration at 40 mmol L-1 with changing AMP 5 concentration (mmol L-1): 20, 30, 40, 50, 60, 70, 80; (iii) Constant AMP and Ag+ concentration at 6 80 and 40 mmol L-1, respectively, with decreasing HNO3 concentration (mmol L-1): 50, 40, 30, 20, 7 10, 5, 0. (C) Enlarged transmission electron microscopy (TEM) image of self-assembled hydrogel 8 structures formed in 40 mmol L-1 AMP/20 mmol L-1 Ag+. Reproduced with permission from ref 9 62. Copyright 2017 Wiley-VCH. 10 41 1 Figure 7. (A) Chemical structures of guanposine and isoguanosine. (B) (i) 2D illustration 2 representing a soft (guanosine: guanosine 5’-monophosphate (Guo:GMP) 1:6) and hard 3 (Guo:GMP 1:1) hydrogel. The diffusion of the cytochrome C particles is represented with yellow 4 arrows. (ii) Methylene blue (MB) release profile from the hydrogel at neutral pH. Different 5 Guo:GMP ratio are plotted with blue (1:6), green (1:2) and red (1:1). (C) Chemical structure of 6 Guo and isoguanosine (isoGuo) hydrogels and scanning electron microscopy (SEM) images of 7 Guo, isoGuo and Guo:isoGuo (1:1). Scale bars of 3 μm. (D) (i) Schematic illustration of 8 supramolecular hydrogel formed when silver ions were used to block the crystallization of the 2’-9 deoxy-fluoroguanosine (FGd). (ii) Microstructures of FGd hydrogel at the concentration of 0.7% in 10 48 N4-octanoyl-2′- deoxycytidine (2’-dCN4-C8) H-bonds, π-π stacking Cross-linked fibrous network Diffusion-mediated release of small molecular weight molecules Injectable Drug delivery Tissue engineering 40 Adenosine-based supramolecular hydrogels Adenine + benzene tricarboxylic acids (1,3,5-tris(4carboxyphenyl)benzene or biphenyl-3,49,5tricarboxylic acid) H-bonds, π-π stacking Good mechanical strength Stimuli-responsive to temperature * Biomedical engineering Drug industry 65,66 Adenine + riboflavin-5’ - phosphate sodium salt H-bonds, π-π stacking Nanofibres produced due to to the chiral nature of the ribityl group of ribofavin-5’-phosphate sodium salt Stimuli responsive to pH, temperature, and shear Hydrogel formed at 30 °C High stiffness * Cell imaging Wound healing Tissue engineering 67 5’-adenosine monophosphate (5AMP) + β-iron oxyhydroxide (βFeOOH) H-bonds, π-π stacking Paramagnetic hydrogel Stimuli-responsive to pH, temperature, and chemical environment * Magnetic resonance imaging Drug delivery Cell labelling Magnetic separation 138 Guanosine-based supramolecular hydrogels 5GMP^23,73 ^23,73 H-bonds, π-π stacking Low life time stability Remove cationic and anionic dyes from aqueous solution * Tissue engineering 78,80 5-GMP + polyamines H-bonds, π-π stacking Improved gelation efficiency * Antitumoral applications Tissue engineering 87 5’-deoxy-5’- iodoguanosine + KB(OH)4 H-bonds, π-π stacking, 5’-deoxy5’-iodoguanosine adopts a syn conformation, enabling intramolecular cyclization Stimuli-responsive to temperature * Drug delivery Tissue engineering 29 Isoguanosine (isoGuo) H-bonds, π-π stacking, monovalent ion coordination (Li+, Na+, K+, Rb+, Cs+) Additional donor and acceptor groups for the outer shell Long-term stability Poor injectability Drug delivery Nanoscopic devices construction Tissue engineering 88 5-GMP H-bonds, π-π stacking, coordination of Fe 3+ and Ca 2+ ) pH-responsive release of doxorubicin * Drug delivery Tissue engineering 89 49 2’-deoxy-2’- fluoroguanosine H-bonds, π-π stacking Addition of Ag+ blocks its crystallization and enhances its stability (> 6 months) * Drug delivery Antibacterial applications Tissue engineering 90 Guo + 5-GMP + KOH H-bonds, π-π stacking 5-GMP improves the solubility of Guo Insolubility of Guo allows gelation of 5-GMP at lower concentrations High negative charge in the hydrogel Stimuli-responsive to pH Different molar ratio of Guo and GMP affects its microscopic and macroscopic properties Injectable Biomolecule encapsulation Tissue engineering 82,93,9 4 Guo + isoGuo + KCl H-bonds, π-π stacking Porous flower-like structures Enhanced stability Injectable Anticancer therapy Tissue engineering 56,95 5-GMP H-bonds, π-π stacking, coordination of trivalent ions (La3+, Ce3+, Eu3+ and Tb3+), iondipole interaction between trivalent ions and G - quartets Stimuli-responsive to pH Denser arrangement of nanofibers induced by La3+ and Ce3 Higher mechanical strength * Electron transport Chiral template Drug delivery Tissue engineering 97 Guo + 8aminoguanosine (8AmG) H-bonds, π-π stacking, coordination of divalent ions (Ba2+, Sr2+ and Pb2+) Greater charge density * Tissue engineering Environmental purposes applications 80 Guo + boric acid + KOH H-bonds, π-π stacking, borate diester bond, coordination of K+ Stability of anionic diesters given by presence of K+ Increased stability Injectable Drug delivery Tissue engineering 39,55 Guo + 2-formylboronic acid + KOH H-bonds, π-π stacking, borate diester bond, coordination of K + Selectivity for K + without interference from other alkali metal ions Injectable Serum K+ detection 100 Guo + 1naphthaleneboronic acid + KOH H-bonds, π-π stacking, borate diester bond, coordination of K+ Stimuli-responsive to pH, glucose and hydrogen peroxide Exhibits better biocompatibility over different cell lines Injectable Printable Drug delivery Tissue engineering 101 Guo + 2-formylboronic acid + tris(2aminoethyl)amine + KCl H-bonds, π-π stacking, iminoboronate bond, coordination of K+ Iminoboronate facilitated hydrogel formation Stimuli-responsive to glucose and pH * Zero-order drug release behavior Tissue engineering 79 Guo + 2-FPBA + KOH + aminoglycoside H-bonds, π-π stacking, iminoboronate bond, coordination of K+ Aminoglycosides work as good hydrogel gelators Injectable Antimicrobial applications in vivo Tissue engineering 77 50 Guo + 2-FPBA + 4-Arm PEG-NH2 H-bonds, π-π stacking, iminoboronate bond, coordination of K+ Stimuli-responsive to pH Self-healing properties Injectable Printable Cancer therapy Tissue engineering 102 Guo + benzene-1,4diboronic acid + KOH H-bonds, π-π stacking, iminoboronate bond, coordination of Mg2+ Increased stability * Tissue engineering 74 Guo + (B(OH)3) + acyclovir + methotrexate + NaOH H-bonds, π-π stacking, acyclovir is inserted into the G-quartet structure and replace Guo molecules, lack of interaction between methotrexate and the hydrogel Wakened hydrogel Stimuli-responsive to glucose, pH, ion concentration Lower mechanical strength Injectable Encapsulation of molecules Controlled drug release Tissue engineering 22 Guo + boric acid + KOH + recombinant human collagen H-bonds, π-π stacking Strong hydrophilicity Stimuli-responsive to pH, light, enzyme activity, ions and temperature * In vivo medical patches Tissue engineering 75 Guo + phenylboronic acid + cetyltrimethylammoniu m bromide + NaOH H-bonds, π-π stacking, electrostatic interactions, hydrophobic interactions Structural modulation by cetyltrimethylammonium bromide concentration * Tissue engineering 103 Guo + arylboronate esters (phenyl boronic acid, 4-nitrophenyl boronic acid) or 4methoxyphenyl boronic acid) + KOH H-bonds, π-π stacking, coordination of K+ Stimuli-responsive to pH Homogenous distribution of cells throughout the gel Doesn’t require additional crosslinking steps postprinting 3D printed Development of in vitro 3D disease models or cell systems Tissue engineering 127 Heterotypic nucleoside-based supramolecular hydrogels Bis-quaternary ammonium gemini surfactant with 5’- uridine monophosphate (5-UMP) + adenosine bases H-bonds, π-π stacking Adenine–uracil mixture leads to aggregation both in the bulk and at the air– water interface Injectable Biosensors 105 Acrylated adenine + acrylated thymine hydrogel + potassium persulfate H-bonds, π-π stacking Reusable adhesion behaviour and tissue adhesive properties in a time-dependent manner Injectable Wound dressing Bio-glues Tissue engineering 110 51 Adenine + uracil into PAAm chains + N,N’- methylene bisacrylamide crosslinker + potassium persulfate H-bonds, π-π stacking Anti-fatigue adhesive and tough hydrogel High mechanical strength provided by covalent bonds of MBA * Wound dressing Bio-glues Cell and tissue adhesives, Biomedical sensors and electrodes Tissue engineering 111 Guanine + Cyd comprising αcyclodextrin, thioketalsmodified guanine and cytosine-terminated PEG H-bonds, π-π stacking Reinforcement effect of H-bonds between Guo and Cyt Introduction of thioketals endowed ROSresponsivity Injectable Drug delivery Anticancer treatment Tissue engineering 114 Guo and Cyt + -3′- (1,2dipalmitoyl-sn-glycero3-phosphate) and + NaCl H-bonds, π-π stacking, coordination of Na+ Controlled release of molecules by both nucleobase and salts Injectable Drug delivery in vivo injection and implantation Tissue engineering 115,116 *The corresponding publications did not give any information on injectability or 3D printability. However, based on the properties of similar formulations, we expect them to be injectable. 52 Corresponding Author M. Godoy-Gallardo, E-mail: [email protected], and R.A. Perez, E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT M.G.-G. has received funding from the postdoctoral fellowship programme Beatriu de Pinós, funded by the Secretary of Universities and Research (Government of Catalonia) and by the Horizon 2020 programme of research and innovation of the European Union under the Marie Sklodowska-Curie grant agreement No 801370. M.A.M.-T. is supported by the Spanish Ministry of Science with the project (RTI2018-096320-B-C21, 2018). R.A.P is supported by the Spanish Ministry by the Ramón y Cajal Program (RYC2018-025977-I) and MINECO/FEDER project (RTI2018-096088-J-100). Additional financial support was provided by the Government of Catalonia (2017 SGR 708). M.G-G and M.M-G contributed equally to this work. ABBREVIATIONS 5-AMP, 5’-adenosine monophosphate; 8AmG, 8-aminoguanosine; FGd , 2’-deoxy-2’- fluoroguanosine; 5-IG, 5-deoxy-5-iodoguanosine; 2-FPBA, 2-formylboronic acid; 5-UMP, 5’- uridine monophosphate; β-iron oxyhydroxide (β-FeOOH); Acv, acyclovir; Ade, adenine; Ado, adenosine; NH4+, ammonium; AFM, atomic force microscopy; Ba2+, barium ion; Ca2+, BDBA, benzene-1,4-diboronic acid; BAs, boric acids; calcium ion; CRL, candida rugose lipase; CTAB, 53 cationic surfactant cetyltrimethylammonium bromide; CD, circular dichroism; Ce3+, cerium ion; CA-Fe3O4 NPs, citric acid-modified iron oxide nanoparticles; CDEX, cyclodextrin; Cyd, cytidine; 5-CMP, cytidine 5’-monophosphate; Cyt, cytosine; M2+, divalent cations; E. coli, Escherichia coli; Eu3+, europium ion; ECM, extracellular matrix; FTIR, fourier transform infrared; diC16-3′-G, G-3′-1,2-dipalmitoyl-sn-glycero-3-phosphate; GSH, glutathione; Gua, guanine; Guo, guanosine; 5-GMP, guanosine 5’-monophosphate; GB, guanosine-borate; G4, guanosine-quadruplex; G-quartets, guanosine quartets; G-ribbons, guanosine ribbons; Fe3+, iron (III) ion; HGF, human gingival fibroblasts; H-bonds, hydrogen bonds; isoGuo, isoguanosine; La3+, lanthanum ion; Li+, lithium ion; LMWG, low molecular weight building blocks; Mg2+, magnesium ion; MBA, N,N’-methylene bis-acrylamide; Hg2+, mercury ion; MTX, methotrexate; MB, methylene blue; NapBA, 1-naphthaleneboronic acid; NHDF, normal human dermal fibroblasts; NLs, nucleotide-based lipid; PBA, phenyl boronic acid; PBS, phosphate buffered saline; Pt2+, platinum ion; p-NPP, p-nitrophenylpalmitate; PAAm, poly(acrylamide); PLA, polylactic acid; KOH, potassium hydroxide; K+, potassium ion; P. aeruginosa , Pseudomonas aeruginosa; C-PEG-C, terminal Cyt-functionalized PEG; PEG, thiolated poly(ethylene glycol); RHC, recombinant human collagen; RP, riboflavin-5’-phosphate sodium salt; SEM, scanning electron microscopy; Ag+, silver; Na+, sodium ion; SCAP, stem cells from the apical papilla; Sr2+, strontium ion; S. aureus, Streptococcus aureus; Tb3+, terbium ion; G-TK-G, thioketalsmodified guanine; TEM, transmission electron microscopy; ThT, thioflavin-T; 3D, three dimensional; Thd, thymidine; Thy, thymine; Et3NH+, triethyl ammonium; TAEA, tris(2aminoethyl)amine; Ura, uracil; Zn2+, zinc. 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