A Review of Applications of Solid-State Nuclear Magnetic Resonance (ssNMR) for the Analysis of Cyclodextrin-Including Systems

Cyclodextrins, cyclic oligosaccharides composed of five or more α-D-glucopyranoside units linked by α-1,4 glycosidic bonds, are widely used both in their native forms as well as the components of more sophisticated materials. Over the last 30 years, solid-state nuclear magnetic resonance (ssNMR) has been used to characterize cyclodextrins (CDs) and CD-including systems, such as host–guest complexes or even more sophisticated macromolecules. In this review, the examples of such studies have been gathered and discussed. Due to the variety of possible ssNMR experiments, the most common approaches have been presented to provide the overview of the strategies employed to characterize those useful materials.


Introduction
Cyclodextrins (CDs) belong to the group of cage molecules; that is, the core of their structure is composed of a hydrophobic cavity that can trap or encapsulate other substances. Those remarkable encapsulation properties lead to a "host-guest" type relationship that can modify or improve the physical, chemical, or biological characteristics of the guest molecule. While the most commonly used CDs are α, β, and γ that contain 6, 7, and 8 glucose subunits, respectively, the large-ring cyclodextrins (LR-CD), composed of nine to more than several hundred units, are also being studied and utilized [1]. Further, apart from native (non-substituted) CDs, their derivatives have found important uses in various fields, such as pharmacy, cosmetics, biomedicine, textiles, and food domain due to their unique properties [2]. In addition to two-component inclusion complexes consisting of a guest molecule and a CD, polymer additives and CDs covalently linked to polymers are also of current interest. The biodegradability, biocompatibility, and versatility of CDs and CD-based materials extend their applications to new areas every year. CDs are commonly used in pharmaceutical formulations, as they increase the solubility of poorly soluble drugs and protect substances against external factors, such as light, humidity, and heat [3]. CDs can mask unpleasant smells or flavors of drugs, which is especially important in formulations dedicated to children [4]. More than 100 original drugs are currently being manufactured with CDs as excipients [5][6][7].
Interactions between CDs (host) and guest molecules may yield a stable complex with a high equilibrium constant. It is not surprising, then, that the number of newly obtained cyclodextrin host-guest complexes is continually increasing. While host-guest CD complexes can be developed as solution-phase formulations, they also have potential benefits as solids. Processes based on co-grinding, kneading, melt-spinning, spray-drying, lyophilization, coprecipitation, and solvent evaporation for the manufacture of CDs complexes have been extensively reported in the literature and in patent publications. In many of those cases, the As expected, the review of the published works ( Table 1) clearly shows that the most common type of the ssNMR experiment applied in the studies of the CD-based materials is the 13 C cross-polarization magic-angle spinning (CP MAS) analysis, which is an obvious choice for the molecular solids and their mixtures. The CP method is based on heteronuclear dipole interactions; therefore, it is sensitive to internuclear distances and molecules mobility and, hence, can be used to monitor the molecular dynamics of complex systems. This method allows for the achievement of high-resolution 13 C NMR spectra of solid materials, which provide information on the chemical and structural features of the samples being studied. To detect changes in crystallinity and the formation of inclusion complexes, the most relevant spectral features to be considered include the evaluation of signal splitting and changes in chemical shifts and linewidths of the detected 13 C NMR signals (Figures 2 and 3). Physical mixtures are often made for comparison to definitively As expected, the review of the published works ( Table 1) clearly shows that the most common type of the ssNMR experiment applied in the studies of the CD-based materials is the 13 C cross-polarization magic-angle spinning (CP MAS) analysis, which is an obvious choice for the molecular solids and their mixtures. The CP method is based on heteronuclear dipole interactions; therefore, it is sensitive to internuclear distances and molecules mobility and, hence, can be used to monitor the molecular dynamics of complex systems. This method allows for the achievement of high-resolution 13 C NMR spectra of solid materials, which provide information on the chemical and structural features of the samples being studied. To detect changes in crystallinity and the formation of inclusion complexes, the most relevant spectral features to be considered include the evaluation of signal splitting and changes in chemical shifts and linewidths of the detected 13 C NMR signals (Figures 2 and 3). Physical mixtures are often made for comparison to definitively confirm the inclusion process as well as to provide the information on the type of interaction between the guest and the host molecules.  13 C-CP/MAS NMR spectra of the crystalline R(+)-α-Lipoic Acid (RALA)-αCD complex, free α-CD, and free RALA, recorded at 6 kHz spinning frequency. The arrows indicate the spinning side bands Adapted from [18], licensed under CC BY 4.0. [18].

CD
Apart from the 'standard' 13C CP MAS experiment in the reviewed literature, successful application of more sophisticated experiments can be found as well. Below, some of those approaches will be summarized, followed by the description of the most interesting cases. Table 1. General information regarding ssNMR application for the examination of the CD-including systems found in the articles published in period 2012-2022. Abbreviations used in the table: SEM (scanning electrine microscopy), XPS (X-ray photoelectron spectroscopy), XRD (X-ray diffraction), PXRD (Powder X-ray diffraction), TGA (thermogravimetric analysis), HR-TEM (high-resolution transmission electron microscopy), HPLC (high-performance liquid chromatography), ATR-FT-IR (attenuated total reflection Fourier-transform infrared spectroscopy), BET (Brunauer-Emmett-Teller method), DTA (differential thermal analysis), DLS (dynamic light scattering), EDS (energy-dispersive X-ray spectroscopy), WAXD (wide-angle X-ray diffraction), SR-FT-IR (synchrotron radiation Fouriertransform infrared spectromicroscopy, EIS (electrochemical impedance spectroscopy), SAXS (smallangle X-ray scattering), ITC (isothermal titration calorimetry), MALDI-TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry).     polymer with poly(l-lactic acid) used as a packaging material, also for medical equipment [80] [101] (e)

Direct Polarization versus Cross Polarization Experiments
CP may seem to be an obvious choice to study the CD-based materials, as it takes advantage of the properties of the protons coupled to the carbons, namely faster relaxation, which allows for reduction of the recycle delay and increases the signal-to-noise intensity in the spectrum at a constrained experimental time. The double irradiation process is used to transfer some of the proton's magnetization to the carbon atoms. However, in complexes with a ligand of low protonation level, such as sodium perfluorooctane [41], perfluorobutyric acid [75], and perfluorooctanoic acid [76], direct polarization (DP) was applied. DP experiments were found to be useful in those cases, as they allowed for an increase in the ratio of guest-to-host signal intensities. In addition, as 13 C CP/MAS NMR relies on the transfer of magnetization from protons or other abundant nuclei to 13 C via the dipolar coupling mechanism, whereas the direct single pulse method does not, the comparison of DP and CP experiments may be very beneficial in some cases, as different information from 13 C CP/MAS and single pulse 13 C MAS spectra can be obtained.
Another type of measurement is the variable contact time (VCT) CP experiment; however, it is rarely applied. In the CD-based materials and complexes, the particular systems may differ in the proton spin-lattice relaxation time in the rotating frame (T 1ρ ) values. Therefore, by recording the 1 H → 13 C CP/MAS NMR spectra with various contact times, it is possible to obtain the collection of spectra in which the relative intensities of signals originating from different phases will vary. This allows one not only to detect the number of phases but also to selectively enhance or suppress the chosen signals, which can help in the signal assignment. VCT experiments found their particular application in the studies of nanosponges without [67] and with a guest [54] (more on nanosponges, please find in Section 4.2 Nanosponges and functionalized CDs). In those cases, not only the structure but also the dynamics of the systems have been analyzed, the latter enabled by the VTC NMR. To quote the already cited ibuprofen study [54]: 'the outcome of VCT data processing is a "dynamic fingerprint" that can be used for the characterization of the polymer-drug system'. The CP-VCT approach is especially useful to compare loaded and not-loaded polymer systems when a structural difference between those two is not significant. This has been used to analyze the CDs which were parts of a polymer in the two studies cited above: CD vs. CD in nanosponges [67] or CD in nanosponges vs. CD in nanosponges with a guest [54].

Nuclei Other Than 13 C
Due to the elemental composition of CDs, the 13C ssNMR experiments are the most common. Far less popular approaches are 1 H NMR [20,28,41,43,61,92,94] and 2 H NMR. The latter is used to analyze in detail a chosen part of a system. The only two examples for the CD-including systems are: CD complex with limaprost [57,58] and CD-including nanochannels for lithium ions [61]. The latter example shows the 2H NMR applicability in systems of high complexity, in this case, composed of polyethylene oxide and α-CDforming tunnels. It should be noted, however, that solid-state spectra for other nuclei, such as 7 Li, 15 N, or 31 P have been also recorded. The choice of the studied nuclei depends on the composition of the guest molecule in the complex. In some cases, the spectra of nuclei other than 13 C or 1 H can be particularly helpful, as they allow one to determine the guest:host ratio in the solid state or detect the presence of various solid phases. This is possible, as the chemical shifts of the nuclei originating from the complexed and "free" guest molecules usually differ.

Relaxation Studies
A relatively often-performed analysis is linking the spin-lattice relaxation times (T 1 ) with the dynamical mobility. For instance, in the already mentioned CD-limaprost study [57,58], where 2H NMR has been used, the T 1 measurements of deuterium atom allowed the conclusion that CD-constrained water molecules mobility is a main factor for the solid-state CD-limaprost complex stabilization. Another example is a temperaturedependent analysis of the CD-polyaniline complex [84], where ssNMR was used to elucidate the inclusion's effect on the structure of polyaniline polymer. T 1 values revealed that the guest's inclusion into the CD's cavity induces acceleration of the twisting motion of polyaniline chain, that the CD inclusion weakens intermolecular π−π interaction, and that this enhances the accompanying twisting motion.

Two-Dimensional Experiments
Structural information on CD-inclusion complexes can be obtained using 1D solidstate NMR experiments that have been applied to studies of crystalline and amorphous CD-based materials. However, in the reviewed literature, singular cases of 2D WISE (two-dimensional WIde-line SEparation), 2D HETCOR (two-dimensional HETeronuclear chemical shift CORrelation experiments), GCOSY (Gradient Correlation SpectroscopY), and GHSQC (Gradient Heteronuclear Single Quantum Coherence Spectroscopy) application are present, the latter two in one article [102]. WISE has been used in cases when an especially high risk of unseparated signals in a spectrum was predicted [56]. The 2D WISE NMR approach was applied for analysis of site-specific dynamics of the CD-including polymers: polyrotaxanes [19]. According to the authors of the study, it proved a dynamic difference between CDs and polyethylene glycol chains as well as the influence of chemical modifications on the polymer side chains.

ssNMR Applicability for Analysis of the Cyclodextrin Complexes
In the previous section, various ssNMR approaches used towards CD-including complexes published within last 10 years have been described. The rest of this review refers to the results of ssNMR application in such systems. This description has been divided into sections on the basis of the reasons for CD's usage. General information has been gathered in Table 1.

Drug Carriers
The application of CDs as drug carriers is the most popular one (Figure 3). In most of the cases, ssNMR is used to prove the complexation, characterize the structure of a formed complex, and find a possible reason for its stability. However, as the usage of CDs serves to enhance the solubility, hence, bioavailability, of medical substances in solid-state forms of medication, a common reason for ssNMR experiments is either differentiation between amorphous and crystalline complex forms [22,23] or comparison of different complexation methods. The examples are the following complexes: CD-bisacodyl, where co-crystallization and co-evaporation have been compared [29], or CD-hydroxytyrosol, where comparison for physical mixing, spray-draying, and freeze-drying methods has been conducted [53]. Another reason to use ssNMR for systems where CD plays a role of a drug carrier is to determine, or at least estimate, the CD-guest ratio, as in case of the CD-sertraline [91] and CD-praziquantel [88] complexes.
An interesting example is the publication regarding the CD-furazolidone complex [56], where all the above-mentioned reasons can be found: thanks to ssNMR, complex formation has been proven, lyophilization has been found more effective than knitting, CD-guest molar ratio has been suggested, and the observed signal-broadening effect pointed out the loss of crystallinity upon complexation.
Another interesting example of the application of ssNMR was the study of the albendazole:succinyl-β-CD inclusion complexes in spray-dried samples [22]. Albendazole itself was found to exist in a few tautomeric forms, depending on its crystal structure. By comparing the chemical shifts present in the spectrum of a complex with the spectra of various forms of albendazole, it was possible to determine the ratio of particular tautomers in the studied complex.

Drug Carriers with Cellulose
A specific group of the CD-including drug carriers form systems with cellulose: CDgrafted cellulose fibers or hydrogels. Their purpose is either to be used as antimicrobial products with controlled drug release or to enhance solubility more than just by complexation with a CD. So far, all the published articles that address CDs grafted on cellulose refer to antimicrobic guests: ciprofloxacin hydrochloride [33], acetylsalicylic acid [36], ketoconazole [37][38][39], ciprofloxacin, doxorubicin, and paclitaxel [25]. Here, except for the mere characterization of the complex's structure, ssNMR's primary use is to prove that a process of CDs' grafting onto cellulose has been successfully completed.

Nanosponges and Functionalized CDs
Nanosponges are polymers, porous nanoparticles used as toxin-removing agents or drug delivery systems [101]. Historically, nanosponges were referred to as 'cyclodextrin nanosponges' because those with CDs were the first ever obtained. Nanosponges are composed of CDs and cross-linking agents. With regard to nanosponges, ssNMR is used to confirm the system's formation, analyze the system's structure, prove the guest's incorporation into CD's cavity, and, as it has been mentioned in Section 2 (ssNMR approaches used to), to differentiate between loaded and not-loaded systems. However, there are more examples. For instance, in [64], the effect of different excipients is measured by the relaxation time of carbons according to the principle that the more mobile the carbon atom, the slower it relaxes; this is, in turn, a plasticizing effect of an excipient. The historical 'cyclodextrin nanosponges' were composed of native CDs. Today, experiments with substituted CDs are performed. For example, in a pH-tunable nanosponge system composed of a cyclodextrin-calixarene mixture, three different CDs (β-CD, methoxy-azido-β-CD, and heptakis-(6-deoxy)-(6-azido)-β-CD) have been tested [81]. ssNMR analysis showed that methylation of CD favors its incorporation in the polymeric material, likely due to an increase in its hydrophobic character.
Not only nanosponges are CD-polymers-based systems; there are also microspheres. As indicated in [103], ssNMR can be used to define the inner structure of such a system. In this particular study, a physical mixture of CD-polymer and cyclosporin A was compared with spherical amorphous solid dispersion of the co-polymer.
Another aspect is research of CD's functionalization, in other words, analysis of changes applied solely to CDs. As an example, it can serve β-CD modification with phosphorus groups. This allows such CDs to serve as promising matrices for environmentally benign fire-resistant coatings [78]. Due to phosphorus presence, for structure verification, 31P ssNMR has been applied. Another interesting case is tosylation of CDs [97]. In this experiment, N-tosylimidazole forms covalent bonds with the CD, and, at the same time, N-tosylimidazole's aromatic ring is incorporated into the CD's cavity. In order to probe spatial proximity between the guest and CD, 1H−1H double-quantum/single-quantum (DQ-SQ) spectroscopy was employed.

Removing Agents and Sensory Devices
While its role as a drug carrier is the CD complex's most common application, the second most common is its role as a removing agent. Such water pollutants are toxins, such as p-nitrophenol [69,70], naphthalene [68], or endocrine-disrupting chemicals, including bisphenol A and other estrogens [30,35,47]. Such systems may be formed out of silica gel CD-including matrices as well [69]. A wide range of ssNMR usage results can be obtained for those systems: from structure characterization and confirmation of the guest incorporation through the differentiation between system's crystallization methods up to 'crystalline or amorphous' statement. In other words, ssNMR enables a complex analysis of a complex itself and delivers information about the complex formation. For example, in one of the p-nitrophenol-removing studies [83], it was shown that the reduced intensity of one of the carbonyls occurred due to esterification of the carbonylic acid of a polyacrylic acid while making a bond with a CD. Moreover, in contrast to sharp ssNMR signals of hydrated CD, broad resonance lines on the spectrum after system's preparation indicate the amorphous character of the generated material.
A thematically separated, but technically similar topic, is CDs' application as sensory devices for ions [20,21,28]. There, ssNMR serves to prove whether a selected ion incorporation took place.

Other Application
CD complexes in solid state are used also as enantioseparators [32], food and flavorencapsulating agents [62,96], as part of the antimicrobial packaging materials [19,46], etc. Thanks to ssNMR, the inner structure, complexation process, CD-guest molar ratio, etc., are defined.
Interesting example are nanoporous materials consisting of CD tunnels, often in the form of polyrotaxanes, where a rotaxane is a mechanically (i.e., without covalent bonds) interlocked molecule consisting of strings and rings (in this case, rings are CDs) [104]. ssNMR experiments of a nanoporous material with different CDs [65] indicate that the material with γ-CD forms a more rigid structure in contrast to other analyzed CDs. There, the splitting of the carbon atom is restricted due to formation of a tunnel-type crystalline system. According to the article published in 2019 [65], this is characteristic of highly cross-linked CDs and polyrotaxanes and can be observed by application of ssNMR.

The Limitations and the Difficulties of ssNMR Approaches in the Studies of CD-Based Materials
Despite all of the advantages and unique opportunities offered by the ssNMR analysis, to maintain the research integrity, the limitations and the difficulties of reviewed approaches in the studies of CD-based materials also must be emphasized. ssNMR is not a perfect tool to study the CD-including systems, as such a method does not exist and, like any other analytical technique, has its disadvantages.
First, the anticipated changes in the ssNMR spectra of the studied materials may not be detectable, or the differences may be small and ambiguous. This can be caused by several reasons, starting from the weakness of interactions between the host and guest molecule, resulting in only slight differences in the chemical shifts between the corresponding atoms of complexed and non-complexed guest molecules. Another reason is associated with the typical composition of the sample. In the 13 C ssNMR spectra of cyclodextrins, both in free and complexed forms, many signals in the wide rage 50-100 ppm can be observed. It is, thus, very likely that the signals originating from the guest molecules may be overlapped with those from the cyclodextrin. This, in some cases, may be solved by setting particular values of either CP contact time or recycle delay to selectively increase the intensity of the signals originating from the guest. However, in most cases, the differences between the relaxation of host and guest are not significant, which hampers such an approach. For example, while studying the loading of γCD-MOF by MTX, the authors have not observed the noticeable shifting of the cyclodextrin signals, explaining that this observation points out that the framework has no strong interactions with the drug molecules [63]. On the other hand, the 13 C NMR data were consistent with the conclusions made by the authors based on the FTIR experiments.
Another aspect is associated with solid state of matter and is often encountered when comparing the ssNMR and NMR analysis in other phases, such as in solutions [105] or supercritical fluids [106]. In the NMR spectra of solution samples, fast molecular motions lead to the averaging of individual contributions to the hamiltonian, whereby only the isotropic value of the chemical shift and J coupling is observed. In the case of samples in solid phase, inhibition of molecular motions leads to restoration of the full hamiltonian of interaction, resulting in a significant increase of the width of signal in the spectrum, often exceeding its spectral range. In order to narrow the line of the NMR spectrum in the solid state, a magic-angle spinning (MAS) is used. It involves rapid rotation of the sample at a specific angle to the external magnetic field. If the angle is approximately 54o, the lines become narrowed due to the physical averaging of the tensor of anisotropy of chemical shift to the isotropic value as well as the reduction of dipole interactions. The probe heads, usually used for measuring in the solid state, allow the rotation with a frequency of 15 kHz; yet, this is often insufficient to completely eliminate the dipolar couplings, particularly in the case of interaction with the hydrogen nuclei. Therefore, during the signal collection, further decoupling of these interactions with the use of high-powered pulses is used.
There are also some technical issues that hinder the application of ssNMR; in general, the relatively high cost of the analysis and long experimental time needed to obtain good quality results, especially in comparison with the FT-IR or PXRD.

Conclusions
Although ssNMR is not the most commonly used analytical method to characterize CDs and CD-based materials, it is likely one of the most versatile and comprehensive. It can be used to study the solid-state materials with all possible long-range order, amorphous, polymorphous, microcrystalline, polycrystalline, or crystalline. In addition, it can be readily applied to study both single-phase materials as well as mixtures and multi-component systems. The number of ssNMR experiments that can be performed for a particular sample is enormous-starting from the most common 13C CP MAS, but, depending on the type of host molecule, analysis of the other nuclei is also possible. Further, due to the non-covalent interactions occurring between the host and guest molecules in the CDs complexes, each of the components usually preserves their particular proton spin-lattice relaxation time in the rotating frame (T1ρ), which provides an opportunity to benefit from the variable contact time CP experiments. Additionally, the variable temperature experiments and relaxation measurements can provide the information on both structure and dynamics of the studied system. Moreover, in some cases, the reaction kinetics, i.e., the activation energy, can be determined on the basis of the ssNMR analysis.
From the practical point of view, ssNMR can be used to confirm the inclusion process, determine the guest:host ratio, assess the number of phases in the solid sample, detect the non-complexed "free" guest and host molecules, and compare the degree of crystallinity. As in the NMR spectra, each signal can be assigned to a particular atom, and it is possible to determine which part of the guest molecule is involved in the formation of an intermolecular force with a CD molecule. In addition, comparison of the chemical shift value originating from the complexed and "free" guest molecule allows it to provide the information on the character of this interaction. Moreover, ssNMR analysis is likely the most convenient way to study how the method of complex preparation affects the structure of the final product.
Due to the limited number of analytical methods that can be applied to study the CDbased materials in the solid state, it is not surprising that the number of works describing successful applications of ssNMR in this field has been increasing constantly for the last 30 years. Assuming that the accessibility of ssNMR analysis is increasing, together with the constant development of this method, it is very likely that soon ssNMR will become a standard approach in the analysis of all types of solid CD-based materials.