**1. Introduction**

Cyclodextrins (CDs) are chemically stable naturally occurring cyclic oligosaccharides consisting of multiple glucose subunits connected by α-1,4 glycosidic bonds [1,2]. There are three main types of CDs that contain six (α-CD), seven (β-CD), and eight (γ-CD) glucose subunits in a ring (Figure 1). These cyclodextrin macromolecules are cone-shaped with a hydrophobic interior cavity and polar exterior surface [3]. Due to their non-toxic nature [1,4–6] and water solubility [7–9], CDs became widely used in various biomedical fields such as drug solubilization [7,10–12], drug delivery [8,13–17], and nucleic acid transfer [18–20].

*Molecules* **2020**, *25*, x FOR PEER REVIEW 2 of 27

**Figure 1.** Chemical structure of (**a**) α-cyclodextrin, (**b**) β-cyclodextrin, and (**c**) γ-cyclodextrin. **Figure 1.** Chemical structure of (**a**) α-cyclodextrin, (**b**) β-cyclodextrin, and (**c**) γ-cyclodextrin.

Compared to other macrocyclic hosts, cyclodextrins are by far the most extensively used in host– guest chemistry applications and medical imaging [21–25]. They tend to be the macrocycle of choice due to their structural advantages and robust ability to form inclusion complexes [21,26]. An inclusion complex is formed when a guest molecule, commonly a small drug, is partially or fully encapsulated inside the host's interior cavity [1,3]. In the case of cyclodextrins, their preferred guest molecules tend to be hydrophobic, making them suitable for binding in the hydrophobic interior. Therefore, cyclodextrins possess the ability to form inclusion complexes with a wide variety of hydrophobic guest molecules [26–28].Formation of inclusion complexes, or molecular encapsulation, can affect the physiochemical properties of the drug or molecule itself, such as solubility and rate of dissolution [3]. CDs are often exploited because of this property in addition to enhancing water solubility of water-insoluble molecules [3]. The exterior of cyclodextrin is predominantly hydrophilic due to the extensive hydrogen bonding network, making it a biocompatible agent for a wide range of applications [1–3,6–17]. These structural factors are largely why CDs are favored when synthesizing inclusion complexes. Electronics and thermodynamics both play a role in determining if a CD will form an inclusion complex with a guest molecule [1]. The driving force for inclusion complexation involves various noncovalent interactions such as desolvation, or removal, of water molecules from the interior cavity and formation of Van der Waals, hydrophobic, and hydrogen bonding interactions [29]. The major driving forces for cyclodextrin complexation are van der Waals interaction and hydrophobic interaction, whereas hydrogen bonding and electrostatic interaction mostly affect the conformation of a particular inclusion complex [29]. CD's ability to form inclusion complexes with small organic molecules has pioneered the field of supramolecular chemistry. More recently, CDs have successfully been used to build various molecular architectures such as catenanes, rotaxanes, pseudorotaxanes, polyrotaxanes, and other molecular machines [30,31]. Compared to other macrocyclic hosts, cyclodextrins are by far the most extensively used in host–guest chemistry applications and medical imaging [21–25]. They tend to be the macrocycle of choice due to their structural advantages and robust ability to form inclusion complexes [21,26]. An inclusion complex is formed when a guest molecule, commonly a small drug, is partially or fully encapsulated inside the host's interior cavity [1,3]. In the case of cyclodextrins, their preferred guest molecules tend to be hydrophobic, making them suitable for binding in the hydrophobic interior. Therefore, cyclodextrins possess the ability to form inclusion complexes with a wide variety of hydrophobic guest molecules [26–28]. Formation of inclusion complexes, or molecular encapsulation, can affect the physiochemical properties of the drug or molecule itself, such as solubility and rate of dissolution [3]. CDs are often exploited because of this property in addition to enhancing water solubility of water-insoluble molecules [3]. The exterior of cyclodextrin is predominantly hydrophilic due to the extensive hydrogen bonding network, making it a biocompatible agent for a wide range of applications [1–3,6–17]. These structural factors are largely why CDs are favored when synthesizing inclusion complexes. Electronics and thermodynamics both play a role in determining if a CD will form an inclusion complex with a guest molecule [1]. The driving force for inclusion complexation involves various noncovalent interactions such as desolvation, or removal, of water molecules from the interior cavity and formation of Van der Waals, hydrophobic, and hydrogen bonding interactions [29]. The major driving forces for cyclodextrin complexation are van der Waals interaction and hydrophobic interaction, whereas hydrogen bonding and electrostatic interaction mostly affect the conformation of a particular inclusion complex [29]. CD's ability to form inclusion complexes with small organic molecules has pioneered the field of supramolecular chemistry. More recently, CDs have successfully been used to build various molecular architectures such as catenanes, rotaxanes, pseudorotaxanes, polyrotaxanes, and other molecular machines [30,31].

During the past few decades, cyclodextrins have become of interest as contrast agents and potential biosensors for different medical imaging modalities [32]. In the context of imaging, CDs have been used primarily as a scaffold to support and/or solubilize smaller molecules that produce or quench a signal for enhanced imaging. However, in a few cases, the unique supramolecular nature of the CD is essential in producing the signal for imaging. Examples of CD-containing constructs that can be imaged by a wide variety of modern imaging technologies are discussed herein. During the past few decades, cyclodextrins have become of interest as contrast agents and potential biosensors for different medical imaging modalities [32]. In the context of imaging, CDs have been used primarily as a scaffold to support and/or solubilize smaller molecules that produce or quench a signal for enhanced imaging. However, in a few cases, the unique supramolecular nature of the CD is essential in producing the signal for imaging. Examples of CD-containing constructs that can be imaged by a wide variety of modern imaging technologies are discussed herein.

Magnetic resonance imaging (MRI) was the first imaging modality to utilize cyclodextrins as contrast agents [33]. CD-based MRI contrast agents produced contrast through the reduction in spinlattice relaxation (T1) time of the water protons. There are two different established methods of synthesis of CD-based MRI contrast agents: (1) host–guest interactions between CD cavity and metal– organic complexes [33,34] and (2) by direct conjugation of CDs to the metal–organic complexes through the external hydroxyl groups of CD molecule [35,36]. The reduction in T1 relaxation for CDbased contrast agents, and therefore their contrast, is substantially stronger compared to the metal– organic complexes on their own [34,37]. Magnetic resonance imaging (MRI) was the first imaging modality to utilize cyclodextrins as contrast agents [33]. CD-based MRI contrast agents produced contrast through the reduction in spin-lattice relaxation (T1) time of the water protons. There are two different established methods of synthesis of CD-based MRI contrast agents: (1) host–guest interactions between CD cavity and metal–organic complexes [33,34] and (2) by direct conjugation of CDs to the metal–organic complexes through the external hydroxyl groups of CD molecule [35,36]. The reduction in T<sup>1</sup> relaxation for CD-based contrast agents, and therefore their contrast, is substantially stronger compared to the metal–organic complexes on their own [34,37].

Secondly, positron emission computed tomography (PET) utilizes CD-based molecular imaging probes [25]. The PET probes emit positron, which annihilates with a stationary electron from the surroundings producing two gamma-photons, which are detected [38]. The PET tracers based on CD Secondly, positron emission computed tomography (PET) utilizes CD-based molecular imaging probes [25]. The PET probes emit positron, which annihilates with a stationary electron from the

can be divided in two classes. The first class contains CD-based nanoparticles (NPs) radiolabeled with

surroundings producing two gamma-photons, which are detected [38]. The PET tracers based on CD can be divided in two classes. The first class contains CD-based nanoparticles (NPs) radiolabeled with either <sup>64</sup>Cu [25] or <sup>18</sup>F [39]. Another recently developed type of CD-based PET imaging agents contains CD molecules conjugated to the p-NCS-benzyl-NODA-GA (NODAGA) chelator labeled with <sup>68</sup>Ga [40,41]. Followed by PET, the CD-based contrast agents were developed for single photon emission computed tomography (SPECT). SPECT contrast agents were created by radiolabeling of CD-based NPs either with 99mTc [42] or <sup>125</sup>I [43].

Recently, CDs were applied as contrast agents for ultrasound (US) and photo-acoustic imaging (PAI). The mechanism of contrast creation for US imaging relies on the substantial differences in the acoustic impedances between the biological tissue and the CD-based agents [44]. The mechanism of PA imaging is more complicated. The PA tracer absorbs the light with subsequent heating. Due to the temperature increase, the contrast agent undergoes thermoelastic expansion resulting in emission of the ultrasonic acoustic waves that can be detected by US receiver [45,46]. The developed CD photoacoustic contrast agents absorbed the light in the infrared range [47–49].

Lastly, multiple studies were conducted to evaluate the performance of the CD-based contrast agents for computed tomography (CT) [50–52]. All of the developments were focused on CD-based NPs that contained metal atoms (Au, Yb, Dy) [50–53]. The presence of the element with high atomic number rises up the X-ray absorption coefficient yielding to the contrast increase. These CD-based contrast agents demonstrated better performance compared to conventional iodine-based CT agents [50,51].

The purpose of this review is to provide an update on recent developments in CD-based contrast agents. A comparison of the developed contrast agents to the clinically available are presented as well as a comparison between different CD-based agents.
