Use of solid-state NMR spectroscopy for investigating polysaccharide-based hydrogels_ A review

Hydrogels find application in many areas of technology and research due to their ability to combine responsiveness and robustness. A detailed understanding of their molecular structure and dynamics (which ultimately underpin their functional properties) is needed for their design to be optimized and these hydrogels to be exploited effectively. In this review, we shed light on the unique capabilities of solid-state NMR spectroscopy to reveal this information in molecular detail. We review recent literature on the advancements in solid-state NMR techniques in resolving the structure, degree of grafting, molecular organization, water-biopolymer interactions and internal dynamical behavior of hydrogels. Among various solid-state NMR techniques, C cross polarization (CP) magic angle spinning (MAS) NMR is examined for its ability to probe the hydrogel and its trapped solvent. Although widely applicable to many types of polymeric and supramolecular hydrogels, the current review focuses on polysaccharide-based hydrogels.


Introduction
Hydrogels are conventionally considered three-dimensional nanofibrous materials consisting of cross-linked hydrophilic polymer networks (Chai, Jiao, & Yu, 2017). They are able to swell and retain large amounts of water, while remaining insoluble and preserving their structural and dimensional constrained integrity due to the presence of chemical or physical cross links (Chivers & Smith, 2019). In addition to covalent cross-linking, the physical cross links can range from entanglements to weak formations of hydrogen bonds, Van der Waals interactions and π-π stacking. Hydrogels are often considered as biocompatible materials, since they possess high water content and a soft nature (Gun'ko, Savina, & Mikhalovsky, 2017). Moreover, in certain implementations they exhibit great similarity to natural extracellular matrices as well as cell adherence surfaces making them a suitable environment for cell proliferation (Dahlmann et al., 2013). Since their discovery and deployment in the biomedical field in the middle of the 20th century, hydrogels have been extensively studied and took a wide share in everyday products, but certain molecular aspects of their behavior and functionality remain incompletely understood (Yahia, 2015).
The most widely known use of NMR spectroscopy is in the liquid or solution state. In the solution state, small soluble molecules experience rapid thermal isotropic motions, which average out all orientation-dependent nuclear magnetic interactions. Then, isotropic components are the only detectable interactions left, resulting in highly resolved solution NMR spectra with excellent signal to noise. Problems arise for molecules that are in an immobilized or "solid" state, where they have restricted motions and are too big to tumble rapidly. This lack of sufficiently rapid isotropic mobility reveals in "solid-state" NMR studies the presence of different types of orientation-dependent nuclear and internuclear interactions (e.g. anisotropic and dipolar interactions) normally hidden in liquid-state NMR of small dissolved molecules. These interactions offer information on local geometric and electronic structure, but on the opposite side, are associated with the loss of resolution, reduced sensitivity and difficulties in the detection of individual atomic sites due to line broadening (Polenova, Gupta, & Goldbourt, 2015). In the absence of line-narrowing techniques (see below), the NMR spectra of most solids are broad and weak, limiting the insights accessible by this technique under such conditions.
Fortunately, several approaches have been developed to regain resolution and sensitivity. To suppress the anisotropic interactions dominating in solid-state, solid-state NMR is often combined with magic angle spinning (MAS). With this approach, the sample is rapidly rotated at an angle of 54.74°with respect to the static magnetic field of the NMR instrument. Undesired line-broadening interactions can be suppressed partially or totally depending on the MAS frequency, with total suppression occurring when the MAS frequency exceeds the magnitude of the interaction (Andrew, Bradbury, & Eades, 1959;Lowe, 1959). The result of this is a ssNMR spectrum with relatively narrow peaks occurring at the same isotropic chemical shift frequencies detected in liquid-state NMR spectroscopy. The use of ever faster MAS has dramatically enhanced the applicability and power of modern ssNMR.
Nowadays, solid-state NMR spectroscopy with its MAS-based techniques has established a firm position in the pharmaceutical and biomedical industry due to its ability to provide detailed molecular information in a nondestructive and noninvasive fashion. MAS NMR yields structural and molecular dynamical information, not only for crystalline structures, but also for amorphous and gel-like environments where other commonly used solid-state techniques have limited capabilities Li et al., 2007;van der Wel, 2017Weingarth & Baldus, 2013).
In Table 1 we summarize a few key differences in the use of solidand liquid-state NMR, which will be further examined in the remainder of this review. Before examining recent applications to polysaccharide hydrogels, we discuss a few more general concerns and how to address them. One downside of spinning at ultra-high frequencies is the creation of frictional heating which can increase the sample temperature by up to 20 K and can be problematic if not compensated with active cooling, especially in case of thermo-responsive hydrogels (Aguilar-Parrilla, Wehrle, Bräunling, & Limbach, 1990;Brus, 2000;Dvinskikh, Castro, & Sandström, 2004;Langer, Schnell, Spiess, & Grimmer, 1999). MAS is often combined with a complementary line-narrowing technique based on the "decoupling" of line-broadening (dipolar) interactions with strong radio-frequency (RF) pulses. These decoupling sequences can also cause substantial sample heating, which is counteracted by additional sample cooling and improved probe designs (Gor'kov et al., 2007;Stringer et al., 2005). Another potential downside of the MAS approach is that it results in the MAS-rate-dependent generation of significant centrifugal forces that can damage sensitive samples (Han et al., 2010;Mandal, Boatz, Wheeler, & van der Wel, 2017;Renault, Shintu, Piotto, & Caldarelli, 2013). The safely achievable spinning frequency and the sample holder (known as MAS rotor) diameter are inversely proportional. Ultra-high spinning frequencies can only be reached with an accompanying reduction of the sample volume. Different types of MAS rotors are shown in Fig. 1 for size comparison. The displayed rotors have outer diameters of 7, 4, 3.2, 1.9 and 1.3 mm, corresponding to maximum internal sample volumes of approximately 240, 71, 30, 13 and 2.5 μL.
Whilst MAS dramatically improves the resolution and signal to noise of ssNMR, further signal enhancement techniques are important to overcome the inherently low sensitivity of the method. This is also connected to the small active volume of the employed MAS rotors (Fig. 1). 13 C cross polarization (CP), which leverages the higher sensitivity and faster relaxation properties of 1 H nuclei, is one means to boost the signal of 13 C (and other less sensitive) nuclei. Moreover, it can be used to provide distinctive information not only on the molecular structure, but also on the molecular interactions, polymorphism, and chemical compositions of the hydrogel. This will be examined in more detail in the papers discussed in this review, which we focus primarily on illustrative recent alginate and chitosan hydrogels. Other noteworthy applications, of especially CP-based ssNMR, are also available on cellulose based materials (Courtenay et al., 2018;Isogai, Usuda, Kato, Uryu, & Atalla, 1989;Kono et al., 2002;Radloff, Boeffel, & Spiess, 1996;Schaefer & Stejskal, 1976;Sparrman et al., 2019). The high rigidity of especially crystalline cellulose makes CP ssNMR especially powerful, as it works optimally in rigid samples (Matlahov & van der Wel, 2018). It has been used to determine the cross-linking degree of superabsorbing networks, probing the network-additives interactions, identifying the solid state structural properties and packing arrangements, characterizing the polymorphic forms and conformational changes affecting the gelation properties (Capitani, Del Nobile, Mensitieri, Sannino, & Segre, 2000;Lenzi et al., 2003;Nonappa &  Kolehmainen, 2016; Ramalhete et al., 2017). Nanochitosan and nanocellulose are of increasing interest in several fields including material science and biomedical engineering. 13 C CP/MAS NMR appears to be of significant importance in identifying the solid state structural properties and packing arrangements of these nanomaterials (Abitbol et al., 2016;Dufresne, 2019;Yang, Wang, Huang, & Hon, 2010). The aim of this review is to examine the importance of solid-state NMR spectroscopy as a versatile analytical technique in resolving the structure and dynamics of a class of biomedical drug and cell delivery hydrogels. Hydrogels are classified as liquids, but behave like solids. The cross-linked nanofibrous network traps water, which in turn expands throughout its spacious volume, forming an insoluble non-fluid gel. These particular properties of a hydrogel make it suitable for use as a decent drug delivery system, allowing an encapsulation (and subsequent release) of target molecules in the trapped water phase. Hydrogels can be formed using different biological and chemical compounds including proteins, amyloid polypeptides, polysaccharides, organic and inorganic polymers (Butcher, Offeddu, & Oyen, 2014;Gibbs, Black, Dawson, & Oreffo, 2016;Naahidi et al., 2017). For biomedical applications bio-compatibility is an important consideration, leading to a particular interest in the development of polysaccharides as biocompatible drug delivery systems (Coviello, Matricardi, & Alhaique, 2006). Based on a combination of practical applications in biomedicine and the illustrative use of incisive ssNMR studies, we focus this review on recent CP MAS ssNMR studies of alginate and chitosan hydrogels.

General properties
Alginate hydrogels show wide applicability as biocompatible materials; their porous structure along with their high water content enables the accommodation of high loads of water-soluble compounds. These properties made them state of the art for use as scaffolds in tissue engineering, vehicles for drug delivery and extracellular matrix models for biological studies (Lee & Mooney, 2012;Tønnesen & Karlsen, 2002).
Alginates are linear polysaccharides with a defined chemical structure assembled from a mixture of two types of monosaccharides (see Fig. 2 and below). Two different types of alginates are well known depending on the source of production: seaweed-derived and bacterial alginate. Seaweed-derived alginate is extracted with an aqueous alkali solution from brown algae (Phaeophyceae), including Ascophyllum nodosum, Macrocystis pyrifera and different Laminaria species. The addition of calcium chloride or other different cationic sources catalyzes the precipitation of the negatively charged alginate, to be followed by an acid treatment to form alginic acid. This production pathway is used in industry for the production of commercial products, due to its simplicity and low production cost (Smidsrød & Skjåk-Braek, 1990). The bacterial production pathway offers more options for tailored chemical structures and physical properties, but is considered more expensive. Bacterial alginate can be produced from Pseudomonas aeruginosa and Azotobacter vinelandii via similar procedures, although this approach yields alginate with G-blocks which form stronger gels with higher viscosity when cross-linked with Ca 2+ ions (Remminghorst & Rehm, 2006;Silva et al., 2012;Urtuvia, Maturana, Acevedo, Peña, & Díaz-Barrera, 2017).

Structure and characterization
D-mannuronate (M) was thought to be the major component of alginate until the L-guluronate (G) subunit was also identified (Fig. 2) (Fischer & Dörfel, 1955). The chemical structure of alginate was distinguished later as series of block copolymers, consecutive G or M residues, and alternating M and G ones. Alginate composes a whole family of unbranched blocks of (1,4) linked β-D-mannuronate and α-Lguluronate residues. The M/G ratios are subjected to natural source variation, hence alginate extracted from different sources will differ in M and G content, length and sequence of the blocks (Gacesa, 1988;Thu et al., 1996). Several chromatographic and spectroscopic techniques were used for the structural analysis and M/G ratio determination of alginate hydrogels such as thin layer chromatography, ion-exchange chromatography, gas chromatography, solution-state NMR, IR, NIR and Raman spectroscopy (Salomonsen, Jensen, Stenbaek, & Engelsen, 2008;Usov, 1999).
Solution-state NMR is a common and extensively used technique, Fig. 1. Solid-state NMR sample sizes. Comparison (left to right) between the size of a 7 mm rotor spinning up to 7 kHz, a 4 mm rotor spinning up to 18 kHz, a 3.2 mm rotor spinning up to 24 kHz, a 1.9 mm rotor spinning up to 42 kHz and a 1.3 mm rotor spinning up to 70 kHz, including their driving caps, and that of a 2€ coin (far left). but acid hydrolysis of the long polysaccharides is often essential for obtaining well-resolved spectra. Partial acid hydrolysis is considered time consuming and is sample destructive. Additionally, broad overlapping lines appear whenever suspended aggregates are present (Grasdalen, 1983). As noted above, in such liquid-state NMR spectra of large slowly tumbling molecules, a combination of chemical shift anisotropy and dipolar interactions, as caused by the reduced mobility of suspended aggregates, are the main causes of broadening. MAS NMR experiments can be used to suppress or overcome the broadening effects associated with these aggregated states. A comparison is shown in Fig. 3a-b between measurements done by 1 H solution-state NMR on hydrolyzed alginate and 1 H MAS NMR on intact alginate samples having various M/G ratios. The resolution and M/G ratios obtained by both techniques are comparable and in good agreement, without the need for hydrolysis for MAS NMR. Upon addition of calcium to the alginate, the negatively charged polysaccharide tends to cross-link into a hydrogel with reduced mobility. As shown in Fig. 3(c-d), upon increasing the calcium content, due to increase in sample viscosity, one observes line broadening and shifting of proton signals in 1 H solution-state NMR spectra. Under such conditions, 1 H MAS NMR is a powerful and alternative technique as the MAS helps suppress these line broadening effects. Thus, MAS NMR on crosslinked hydrogels can be useful to resolve the structure and determine the M/G ratios, especially as it bypasses the need for destructive and time-consuming acid hydrolysis procedures (Salomonsen, Jensen, Larsen, Steuernagel, & Engelsen, 2009a). 13 C CP/MAS NMR spectroscopy represents an additional technique that provides insight at the atomic-resolution level into alginate hydrogel structure and dynamics. Unlike the 1 H NMR mentioned above, here one detects the signal of 13 C isotopes present in the hydrogels (often at natural abundance). It is important to note that the peak intensities in 13 C CP/MAS NMR spectra are not usually quantitative measures. The reason for this is that the efficiency of CP-based magnetization transfer from 1 H to 13 C depends on the strength of the dipolar interaction between 1 H and 13 C, which varies across the molecule and depends on local mobility (Matlahov & van der Wel, 2018). That said, various techniques have been developed that much improve the reliability of quantitative interpretation of CP/MAS NMR (Hou et al., 2006;Johnson & Schmidt-Rohr, 2014;Takeda et al., 2012). Moreover, relative changes in peak intensity can be used to obtain valuable (semi) quantitative insights into the composition of different samples with similar characteristics. The 13 C CP/MAS NMR spectra in Fig. 4 show the anomeric carbons around 101 ppm and the ring carbons in the range of 60−90 ppm (Mollica, Ziarelli, Lack, Brunel, & Viel, 2012;Salomonsen, Jensen, Larsen, Steuernagel, & Engelsen, 2009b;Sperger, Fu, Block, & Munson, 2011). Assignments of the alginate peaks from 13 C CP/MAS NMR (Salomonsen et al., 2009a) are shown in Table 2. Since solution and solid-state MAS NMR chemical shifts are directly comparable, assignments of the observed NMR signals are often performed with the support from 13 C solution-state NMR data. Similarly, M/G ratios are calculated and structural changes can be directly detected. However, the perturbation in the chemical shifts of neighboring residues often cannot be identified in the 13 C CP/MAS NMR spectra due to limited resolution.
It is worth noting that solid-state NMR generally suffers from a reduced resolution relative to typical high-resolution solution NMR data. This can stem from various sources, including the presence of structural heterogeneity, specific time scales and modes of dynamics, and limiting hardware specifications (including field strength and MAS rate). One of the key advances in modern solid-state NMR stems from an increased awareness of the controllable parameters that can improve the resolution. One key finding that is quite appreciable in biomolecular solidstate NMR (Mandal et al., 2017;Marassi & Crowell, 2003;Martin & Zilm, 2003), is that presence of optimized levels of hydration can be highly beneficial. Hydrated samples display increased mobility, which can both help and hurt the spectral resolution, depending on the timescale of motion. The dynamics (and thus resolution) may be tuned to some degree by modulating the solvent coupled dynamics based on changes in viscosity and sample temperature, among others (Li et al., 2019;Mandal et al., 2015;Sarkar et al., 2016). Complementing these sample optimization approaches, modern solid-state NMR can also offer improved resolution by increased access to high-field NMR instrumentation, ultrafast MAS equipment and new pulse sequence developments, facilitated in part by access to national and international shared facilities.

Table 2
Assignments of the 13 C CP/MAS NMR peaks of D-mannuronic acid and L-guluronic acid in Fig. 4. (Salomonsen et al., 2009a Fig. 5). Broadening affects specific signals such as signals of carbonyl units ranging from 170 to 180 ppm and M unit pyranose rings around 75 ppm, undoubtedly indicating a distinct divergence in the local structures of the alginate hydrogels. A systematic relation was also observed between the increase in broadening of M signals and the decrease in the polyvalent ion size (Brus et al., 2017;Urbanova et al., 2019). The broadening and disappearance of the M4 and M5 carbon signals, while G units are substantially unaffected, point to a degree of conformational diversity of the MG, MM, and GG blocks, their interaction with the polyvalent cations and their role in forming stable complexes (Agulhon, Robitzer, David, & Quignard, 2012;Hecht & Srebnik, 2016). Disregarding the different cationic species used for gel cross linking, the relatively narrow 13 C CP/MAS NMR signals reflect the uniformity of G-rich blocks in the measured hydrogels. The GM and MM blocks are expected to have a more open geometry than GG ones, thus further depending on the size, valence and affinity of interacting cationic species. Therefore, accepting more interchain aggregation and conformational flexibility. Alginate polymer chains are known to be rigid, but a certain degree of internal motion exists in strongly hydrated domains. The broadening observed in the 13 C CP/MAS NMR signals in Fig. 5 could be associated with the presence of segmental dynamics (Brus et al., 2017;Urbanova et al., 2019). Further solid-state NMR studies of these structural and motional aspects of these polysaccharide hydrogels are needed for a specific analysis and interpretation of these molecular features. Fortunately, solid-state NMR offers an array of complementary approaches that probe the local and overall dynamics of the system (Matlahov & van der Wel, 2018).

Local environment diversity
MAS NMR can also provide a direct view of the cross-links themselves. To investigate deeply the local environment near Al 3+ ions in alginate gels, 2D 27 Al triple quantum (TQ) MAS NMR experiments have been used (Brus et al., 2017;Urbanova et al., 2019). Two distinct Al 3+ sites appeared clearly at 0.3 and 1.4 ppm shown in Fig. 6. Moreover, the 2D NMR analysis revealed that the octahedral coordination structure of Al 3+ ions in the two sites showed distinct quadrupole coupling constants (C q ) of 4.0 and 2.6 MHz, respectively. That difference revealed the local coordination geometry for the site with C q =2.6 MHz to have a spherical symmetry, while the other site has significant distortions in the local symmetry. The two types of cross linking centers have comparable population, determined from the signal intensities. The motion of the water molecules and mobility of the central ions were found to be significant factors affecting the local dynamics (Brus et al., 2017;Urbanova et al., 2019).

General properties
Chitosan is a linear polysaccharide produced commercially by chemical modification (deacetylation) of chitin. Chitin is a celluloselike biopolymer that is mainly found in the exoskeleton of aquatic marine animals such as shrimps, crabs, and lobsters, and cell walls of fungi. NMR spectroscopy is the most reliable technique to determine the degree of deacetylation, which ranges between 60-100 % for commercially available material. Chitosan was proposed as a promising  The top shows the full 1D projection (black) and 1D slices extracted from the two observed Al 3+ sites (red, blue). Adapted with permission from (Brus et al., 2017) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). candidate for therapeutic applications and wound healing because of its properties as a cholesterol trapping agent, antioxidant, antibacterial and its hypoglycemic activity in the prevention of chronic diseases. The versatility of chitosan promoted its usage in waste management, agriculture, water purification, cosmetics, dentistry, food packaging and drug delivery systems (Hamedi, Moradi, Hudson, & Tonelli, 2018;Shariatinia, 2018).
3.2. Structure and degree of grafting 13 C CP/MAS NMR methods have proved useful for probing the chemical and structural conversions of chitosan and related biomassderived materials. The technique has been used to evaluate the ability of thermal treatments to change the normally amorphous nature of carboxymethyl chitosan (CMC). All structures resulting from thermal treatment in 13 C CP/MAS spectra showed broad signals indicative of a wide distribution of local structures (Capitani, De Angelis, Crescenzi, Masci, & Segre, 2001). Therefore, thermal treatment did not result in a transformation of the amorphous structure of chitosan into a long-range crystalline defined one. The dynamics of the system could also be monitored by 13 C CP/MAS dynamic experiments. The solid-state NMR measurements determined the dipolar coupling between directly bonded 1 H and 13 C sites, based on the optimal CP contact time (0.5 and 0.4 ms for untreated and treated CMC samples, respectively) where the maximum signal to noise occurs in the 13 C spectra. As this coupling parameter is sensitive to dynamic averaging (manifest in reduced order parameters), its measurement by CP/MAS NMR can be used to probe local mobility. All values obtained for CMC fall in the very rigid range of values, representing relatively high dipolar coupling order parameters, although with a slight increase in order for the thermally treated sample (Di Colo et al., 2006). 13 C direct excitation, sometimes known as direct polarization, MAS 13 C NMR spectra provide a complementary method of measuring 13 C signals by MAS NMR. Under the right conditions, these direct-excitation experiments permit a more quantitative analysis than is achievable by standard CP-based MAS NMR (Hou et al., 2006;Kono & Teshirogi, 2015). This is due to the fact that these experiments are not reliant on 1 H-13 C dipolar couplings to polarize the 13 C signal (see also above).
Such data are shown in Fig. 7 for cyclodextrin-grafted carboxymethyl chitosan (CD-g-CMC) and CMC hydrogels (Kono & Teshirogi, 2015). In these CMC samples, cyclodextrin (CD) units are attached to a subset of the CMC monosaccharide building blocks in order to facilitate the absorption properties toward acetylsalicylic acid (Aspirin), thus obtaining a biodegradable material possessing controlled on-demand drug release ability. These MAS NMR data provide significant structural information including 13 C resonance assignments and degree of grafting for carboxymethyl cyclodextran (CMCD). Overlap of the 13 C resonance peaks between CMC and those of CMCD occurs due to the chemical similarity and thus similarity of many of the chemical shifts. However, two peaks of CMC in the region of 52−58 ppm and 20−24 ppm, assigned to the C2 and the acetamide CH 3 group, can be distinguished separately from the peaks of CMCD. Additionally, several peaks in the range of 170−182 ppm region can be distinguished. The resonance at 178 ppm was assigned to carboxylate carbonyl carbons, while the one at 172 ppm was for amide carbonyl carbons and acetamide groups. Thus, by monitoring the appearance of these characteristic signals, MAS NMR allows for the detection of the incremental degrees of CD grafting. The graft degree of CMCD is considered as the average number of grafted CMCD per one monomer unit of CMC. The structural parameters obtained for each sample revealed clearly that an increase in the feeding ratio of CMCD to CMCs during the CD-g-CMC preparation procedure is followed by an increase in the degree of CD grafting in the gel network (Kono & Teshirogi, 2015;Kono, Onishi, & Nakamura, 2013;Kono, 2014).

Dynamic behavior of water molecules
Another valuable use of solid-state NMR is in the study of solvent interactions and solvent mobility within the hydrated hydrogels. The hydration characteristics of hydrogels are important for the mechanical and functional properties that are relevant for many applications. Variable temperature 2 H static NMR was previously used to determine the different water species in hydrated chitosan. Mobility of the water molecules is a major factor affecting the broadness of the 2 H NMR peaks such that: rigid components having restriction in mobility experience strong quadrupole interactions, thus leading to a broad peak. Meanwhile, mobile components having more freedom in mobility express weak quadrupole interactions, thus leading to a narrow peak. At room temperature, the broad peak of the rigid 2 H component is assigned to deuterons present as ND/OD, which experience rapid exchange with D 2 O. The narrow peak of the mobile 2 H component is assigned to the weakly bound and free water, which experience higher mobility upon temperature increase. Upon decreasing the temperature to 190 K, the study observed coexistence of strongly bounded water to the biopolymer matrix, in a rigid amorphous form, non-freezable water exhibiting high mobility and flipping water that are immobilized and are able to undergo a 180°flip similar to crystalline hydrates. Therefore, four water species shown in Fig. 8 were identified: free water experiencing unrestricted motion, highly mobile but weakly bound Fig. 7. Quantitative MAS NMR analysis of modified chitosan hydrogels. 13 C direct excitation MAS NMR spectra of CMC hydrogel and CD-g-CMC hydrogels. The spectra are normalized to the methyl carbon peak intensity at 22 ppm. 1 H-13 C dipolar decoupling was applied to enhance resolution. Adapted with permission from (Kono & Teshirogi, 2015). water, rigid non-freezable matrix waters, and flipping water.
Although the solid-state NMR studies above are applied to polysaccharide samples in which the molecules are present randomly in all possible orientations (a powder distribution), solid-state NMR studies can also be applied to (partly) oriented or aligned samples. For example, compared to completely random orientations of deuterons present as ND/OD in a normal powder, alignment along the magic angle in static mode yields distinct NMR spectra. This method of preparing the sample has allowed insight into the structuring and orientation of the polymeric chain, for example in presence and absence of stretching forces. Thus information can be obtained via solid-state NMR that examines the important role of solvation water on the toughness, structure, and material properties of biomaterials (Radloff et al., 1996;Wang, Zhang, Chen, & Sun, 2016).

Conclusions and future perspectives
Various models and theories have been presented and enormous efforts have been made to understand the interpenetrating network, packing and mobility of hydrogels, but still limitations exist and controversies are not uncommon. Our understanding of the structural and chemical aggregation transformations, and water-matrix interaction pathways occurring in hydrogels is still limited (Hoffman, 2012). Fundamental progress in this direction can pave the way for designing the next generation of polysaccharide hydrogels. As we have seen, solid-state NMR spectroscopy is a promising technique in resolving asyet missing aspects of the molecular structure, polymorphism, packing and dynamics of hydrogels. This is true for the systems examined above, as well as other nano-polysaccharides such as nanocellulose e.g. (nanofibrils and nanocrystals) and nanochitosan. These bio-derived materials may enable impressive and promising applications in different fields, which however require a deeper understanding of their molecular underpinnings. As we have tried to emphasize, one strength of modern solid-state NMR is its diversity of methods and ability to reveal many different aspects of molecular structure, dynamics and interactions. It is important to note that the work discussed above is just a modest, but hopefully informative, sampling of an ever growing literature.
There is substantial reason to be further optimistic about an even greater expansion of capabilities for the studying polysaccharide hydrogels. The reason for this is the ongoing advancements in solid-state NMR instrumentations and techniques, inspired by the world of biomolecular ssNMR and studies of non-hydrogel materials. Dramatic improvements in sensitivity can be gained by combining novel techniques such as ultrafast MAS (at rates exceeding 100 kHz) as well as dynamic nuclear polarization (DNP). Spectroscopic sensitivity is a critical parameter upon studying nanofibrous interpenetrating systems. DNP permits large signal enhancements overcoming sensitivity limitations, however also requires very low temperatures which could affect the behavior of the hydrogel Koers et al., 2014;Mance et al., 2017;Medeiros-Silva et al., 2018;Smith et al., 2018). One exciting aspect of ultrafast MAS probeheads is that they can enable 1 Hdetection with and without deuteration (Baker et al., 2018;Mance et al., 2015). An important factor in biomolecular ssNMR is the use of advanced isotopic labelling approaches (Baker & Baldus, 2014;Baker, Daniëls, van der Cruijsen, Folkers, & Baldus, 2015;van Zandvoort et al., 2015), which may also be valuable in future studies of polysaccharide hydrogels. An essential part of optimal use of solid-state NMR will be the pursuit of integrated methods, such as the combination of experimental solid-state NMR with ever-improving computational approaches (Rad-Malekshahi et al., 2015;Weingarth et al., 2012), and microscopic analysis such as Atom Probe Tomography and Cryo-Electron Tomography (Baker et al., 2018;Schmidt et al., 2018). Thus, based on the current literature, there is ample room for the increased application of the power and versatility of modern solid-state NMR to elucidate important features of responsive and non-responsive hydrogels. As illustrated by the work discussed in the current article, and the much broader solid-state NMR literature, great opportunities are available to learn about the structure, cross-linking, packing and mobility of interpenetrating gel networks formed by polysaccharides and other biopolymers alike.