2.3.1. Hyperpolarized <sup>13</sup>C CD-Based Contrast Agents

Due to the high signal enhancement, molecular imaging using HP <sup>13</sup>C-containing molecule becomes possible. β-CD was used to create a contrast for HP <sup>13</sup>C MRI [86]. Keshari et al. showed that the host–guest interaction between a β-CD cavity and HP benzoic acid substantially decrease the T<sup>1</sup> relaxation of both HP <sup>13</sup>C nuclei resulting in negative image contrast (decrease in signal intensity) induced by the presence of supramolecular cage (Figure 7). Based on this result, the authors proposed that similar mechanism of the negative contrast can be used to study the interaction of ligand–receptor pairs in vivo. *Molecules* **2020**, *25*, x FOR PEER REVIEW 11 of 27 relaxation of both HP 13C nuclei resulting in negative image contrast (decrease in signal intensity) induced by the presence of supramolecular cage (Figure 7). Based on this result, the authors proposed that similar mechanism of the negative contrast can be used to study the interaction of ligand– receptor pairs in vivo.

**Figure 7.** In vitro experiment at 14T demonstrating the potential application of β-CD as a contrast agent for hyperpolarized (HP) 13C MRI. (**a**) Proton gradient echo image demonstrating the position of phantoms with a different concentration of β-CD (0–10 mM). The HP 13C imaging was performed after the administration of 2.5 mM HP [1-13C] benzoic acid. It can be seen that the MRI signal decreases with β-CD concentration. (**b**) Relative MRI 13C signal dependence on β-CD concentration [86]. The images are reprinted with permission from publisher [86]. **Figure 7.** In vitro experiment at 14T demonstrating the potential application of β-CD as a contrast agent for hyperpolarized (HP) <sup>13</sup>C MRI. (**a**) Proton gradient echo image demonstrating the position of phantoms with a different concentration of β-CD (0–10 mM). The HP <sup>13</sup>C imaging was performed after the administration of 2.5 mM HP [1-13C] benzoic acid. It can be seen that the MRI signal decreases with β-CD concentration. (**b**) Relative MRI <sup>13</sup>C signal dependence on β-CD concentration [86]. The images are reprinted with permission from publisher [86].

DNP was used to create a CD contrast agent for DNP HP MRI [87]. Caracciolo et al. observed the polarization level of 10%. Unfortunately, the T1 relaxation of β-CD protons was equal to 1s at 300 K, which made HP β-CD inapplicable for molecular imaging purposes. On the other hand, HP β-CD can be of interest in the fields, which require the production of strong 1H NMR signal from CD molecules [87]. Following this work was the hyperpolarization of methylated β-CD [88]. The methylation has been conducted using 13CH3I, which enriched the potential contrast agent with 13C and 1H nuclei. Methylated β-CDs underwent DNP and polarization levels of 7.5 and 7% were achieved for 1H and 13C, respectively. The proton T1 relaxation times were found to be similar to those published in [87]; however, the T1 relaxation time of 13C nuclei was equal to 3.3 and 4.9 s for fully methylated β-CDs and partially methylated β-CDs, respectively. These relaxation times allow further DNP was used to create a CD contrast agent for DNP HP MRI [87]. Caracciolo et al. observed the polarization level of 10%. Unfortunately, the T<sup>1</sup> relaxation of β-CD protons was equal to 1s at 300 K, which made HP β-CD inapplicable for molecular imaging purposes. On the other hand, HP β-CD can be of interest in the fields, which require the production of strong <sup>1</sup>H NMR signal from CD molecules [87]. Following this work was the hyperpolarization of methylated β-CD [88]. The methylation has been conducted using <sup>13</sup>CH3I, which enriched the potential contrast agent with <sup>13</sup>C and <sup>1</sup>H nuclei. Methylated β-CDs underwent DNP and polarization levels of 7.5 and 7% were achieved for <sup>1</sup>H and <sup>13</sup>C, respectively. The proton T<sup>1</sup> relaxation times were found to be similar to those published in [87]; however, the T<sup>1</sup> relaxation time of <sup>13</sup>C nuclei was equal to 3.3 and 4.9 s for fully methylated β-CDs and partially methylated β-CDs, respectively. These relaxation times allow further application of HP β-CD as contrast agents in the molecular imaging field. In addition, authors demonstrated the method of further increasing relaxation times [88].

#### demonstrated the method of further increasing relaxation times [88]. 2.3.2. CD-Based Molecular Probes for Hyperpolarized <sup>129</sup>Xe MRI

widely used in the field of molecular imaging using HP 129Xe.

2.3.2. CD-Based Molecular Probes for Hyperpolarized 129Xe MRI Current studies of molecular imaging with HP 129Xe MRI utilizes hyperpolarized chemical exchange saturation transfer (HyperCEST) [89,90]. The HyperCEST effect relies on a constant chemical exchange between the dissolved HP 129Xe nuclei in the solution and supramolecular host Current studies of molecular imaging with HP <sup>129</sup>Xe MRI utilizes hyperpolarized chemical exchange saturation transfer (HyperCEST) [89,90]. The HyperCEST effect relies on a constant chemical exchange between the dissolved HP <sup>129</sup>Xe nuclei in the solution and supramolecular host that can effectively encapsulate <sup>129</sup>Xe [79]. Following selective depolarization of the <sup>129</sup>Xe nuclei encapsulated in the supramolecular cage, the decrease in the dissolved phase <sup>129</sup>Xe MRI signal can be observed as a result of the exchange dynamic [79,89].

application of HP β-CD as contrast agents in the molecular imaging field. In addition, authors

that can effectively encapsulate 129Xe [79]. Following selective depolarization of the 129Xe nuclei encapsulated in the supramolecular cage, the decrease in the dissolved phase 129Xe MRI signal can be observed as a result of the exchange dynamic [79,89]. The interaction between HP 129Xe and the α-CD cavity was studied for the first time in 1997 [91]. The interaction between HP <sup>129</sup>Xe and the α-CD cavity was studied for the first time in 1997 [91]. Authors observed that spin polarization induced nuclear Overhauser effect (SPINOE) and related transfer of nuclear polarization to the α-CD protons [91]. However, this approach did not become widely used in the field of molecular imaging using HP <sup>129</sup>Xe.

Authors observed that spin polarization induced nuclear Overhauser effect (SPINOE) and related transfer of nuclear polarization to the α-CD protons [91]. However, this approach did not become CD-based contrast agents for HyperCEST molecular imaging became of interest recently. The first detection of HyperCEST effect using α-CD-based molecules was achieved from the

observed HyperCEST depletion was equal to 30%, which is significantly smaller compared to other molecular imaging probes [89,93]. The first β-CD-based molecular imaging probe for HyperCEST detection has been developed recently [94]. The HyperCEST contrast agent was realized as cucurbit[6]uril-based rotaxane in which β-CDs played the role of a stopper. Although rotaxane interaction with 129Xe was through cucurbit[6]uril cavity and no HyperCEST effect was observed from β-CDs, the presence of β-CDs was required in order to increase the solubility of the end groups [94].

CD-based contrast agents for HyperCEST molecular imaging became of interest recently. The

α-CD pseudorotaxane complex with five carbon diethylimidazolium bar in aqueous solution [92]. The observed HyperCEST depletion was equal to 30%, which is significantly smaller compared to other molecular imaging probes [89,93]. The first β-CD-based molecular imaging probe for HyperCEST detection has been developed recently [94]. The HyperCEST contrast agent was realized as cucurbit[6]uril-based rotaxane in which β-CDs played the role of a stopper. Although rotaxane interaction with <sup>129</sup>Xe was through cucurbit[6]uril cavity and no HyperCEST effect was observed from β-CDs, the presence of β-CDs was required in order to increase the solubility of the end groups [94]. *Molecules* **2020**, *25*, x FOR PEER REVIEW 12 of 27

Finally, the HyperCEST effect from γ-CD-based pseudorotaxane was observed after the complexation of γ-CD with bisimidazolium guest [95]. Although γ-CD cavity is too large to sufficiently interact with HP <sup>129</sup>Xe, the cavity size can be decreased by threading it with a long alkyl chain. Threading these guest molecules through the cavity of γ-CD reduces the cavity size in order for adequate HP <sup>129</sup>Xe binding to occur, thus making it suitable for HyperCEST detection (Figure 8a). Potentially, this same concept can be applied for any supramolecular cage with a reasonably large cavity. The HyperCEST effect detected from γ-CD-based pseudorotaxanes was equal to 47.5% on average, which makes them interesting candidates for further application in vivo [95]. The main advantage of the HyperCEST contrast agents based on pseudorotaxane architecture over the other studied HP <sup>129</sup>Xe hosts is the ease synthesis and of functionalization of the pseudorotaxanes. Finally, the HyperCEST effect from γ-CD-based pseudorotaxane was observed after the complexation of γ-CD with bisimidazolium guest [95]. Although γ-CD cavity is too large to sufficiently interact with HP 129Xe, the cavity size can be decreased by threading it with a long alkyl chain. Threading these guest molecules through the cavity of γ-CD reduces the cavity size in order for adequate HP 129Xe binding to occur, thus making it suitable for HyperCEST detection (Figure 8a). Potentially, this same concept can be applied for any supramolecular cage with a reasonably large cavity. The HyperCEST effect detected from γ-CD-based pseudorotaxanes was equal to 47.5% on average, which makes them interesting candidates for further application in vivo [95]*.* The main advantage of the HyperCEST contrast agents based on pseudorotaxane architecture over the other studied HP 129Xe hosts is the ease synthesis and of functionalization of the pseudorotaxanes.

**Figure 8.** (**a**) Schematic representation of how CD-based ternary complexes are formed in the presence of HP 129Xe. The guest is threaded through the hydrophobic cavity of cyclodextrin and HP 129Xe is introduced. Detection via HyperCEST is obtained in order to determine if HP 129Xe is bound in the cavity of CD. The images are reprinted with permission from publisher [95]. (**b**) The developed cyclodextrin-based pseudorotaxane used for in vitro HyperCEST detection [95]. **Figure 8.** (**a**) Schematic representation of how CD-based ternary complexes are formed in the presence of HP <sup>129</sup>Xe. The guest is threaded through the hydrophobic cavity of cyclodextrin and HP <sup>129</sup>Xe is introduced. Detection via HyperCEST is obtained in order to determine if HP <sup>129</sup>Xe is bound in the cavity of CD. The images are reprinted with permission from publisher [95]. (**b**) The developed cyclodextrin-based pseudorotaxane used for in vitro HyperCEST detection [95].

In addition, γ-CD pseudorotaxane complexes prove to be a sufficient paradigm for HP 129Xe MRI. The synthesis of the threads proceeds in one step and can be functionalized with different In addition, γ-CD pseudorotaxane complexes prove to be a sufficient paradigm for HP <sup>129</sup>Xe MRI. The synthesis of the threads proceeds in one step and can be functionalized with different terminal

terminal end groups as well as different chain lengths. The 8- and 10-carbon alkyl chains were

maximum HyperCEST depletion for γ-CD pseudorotaxanes was present when the samples were irradiated at a frequency of +128 ppm [95]. γ-CD pseudorotaxanes exhibited binding on the order of 103 at 1:1 host–guest complexation, proving to be sufficient association for HP 129Xe. In addition, binding proved to be two magnitudes of order lower in fetal bovine serum, indicating that this system

would work sufficiently as a potential biosensor in vivo [95].

end groups as well as different chain lengths. The 8- and 10-carbon alkyl chains were functionalized with ethylimidazoliums (Figure 8b), strategically used to enhance water solubility of the inherently hydrophobic alkyl chain [95]. The γ-CD pseudorotaxane was comparable to that of CB6, a known xenon cage that has been responsive to in vivo HP <sup>129</sup>Xe MRI [80]. Similar to CB6, the maximum HyperCEST depletion for γ-CD pseudorotaxanes was present when the samples were irradiated at a frequency of +128 ppm [95]. γ-CD pseudorotaxanes exhibited binding on the order of 10<sup>3</sup> at 1:1 host–guest complexation, proving to be sufficient association for HP <sup>129</sup>Xe. In addition, binding proved to be two magnitudes of order lower in fetal bovine serum, indicating that this system would work sufficiently as a potential biosensor in vivo [95]. *Molecules* **2020**, *25*, x FOR PEER REVIEW 13 of 27

#### **3. CDs-Based Contrast Agent for Ultrasound Imaging and Photoacoustic Imaging 3. CDs-Based Contrast Agent for Ultrasound Imaging and Photoacoustic Imaging**

In addition to MRI contrast agents, CDs have been used for ultrasound (US) imaging. The first approach of synthetizing the potential contrast agent was developed by Cavalieri et al., in 2006 [96]. Air-filled polymer microbubbles functionalized with β-CDs were used as a source for contrast. The contrast originates from significant difference between acoustic impedance of tissue and air encapsulated inside of a microbubble. Cavalieri et al. found that conjugating microbubbles to β-CD preserves them from random coil to α- helix conformation transition. In addition, due to the presence of β-CD, these contrasts allow hosting molecules with hydrophobic features [96]. In addition to MRI contrast agents, CDs have been used for ultrasound (US) imaging. The first approach of synthetizing the potential contrast agent was developed by Cavalieri et al., in 2006 [96]. Air-filled polymer microbubbles functionalized with β-CDs were used as a source for contrast. The contrast originates from significant difference between acoustic impedance of tissue and air encapsulated inside of a microbubble. Cavalieri et al. found that conjugating microbubbles to β-CD preserves them from random coil to α- helix conformation transition. In addition, due to the presence of β-CD, these contrasts allow hosting molecules with hydrophobic features [96].

After nearly a decade of inactivity, the second attempt of using a β-CD-based contrast agent for US imaging was made in 2015 [44]. The authors demonstrated use of a perfluorinated FC-77/ β-CD complex. A visible blurring of signal from FC-77/β-CD caused by a disruption of the inclusion complexes under US was detected [44]. After nearly a decade of inactivity, the second attempt of using a β-CD-based contrast agent for US imaging was made in 2015 [44]. The authors demonstrated use of a perfluorinated FC-77/ β-CD complex. A visible blurring of signal from FC-77/β-CD caused by a disruption of the inclusion complexes under US was detected [44].

Another US medical imaging modality that utilized CD-based contrast agents was PAI. The first CD-based contrast agent was synthetized by surface modification of oleic-acid (OA) stabilized upconversional NPs (UCNPs) NaYF4:Yb3+, Er3<sup>+</sup> with α-CD [49]. α-CD formed an inclusion complexes with an OA yielding to luminescence quenching of UCNPs and production of a strong PA signal instead (Figure 9a). Cytotoxicity studies demonstrated no toxicity of α-CD/UCNPs contrast agents. Following in vitro imaging (Figure 9b), the first PAI in a living mouse was conducted using 980 nm excitation laser. These results demonstrated the ability of α-CD/UCNPs to be an efficient PAI contrast agent for diagnostic purposes [49]. Another US medical imaging modality that utilized CD-based contrast agents was PAI. The first CD-based contrast agent was synthetized by surface modification of oleic-acid (OA) stabilized upconversional NPs (UCNPs) NaYF4:Yb3+, Er3+ with α-CD [49]. α-CD formed an inclusion complexes with an OA yielding to luminescence quenching of UCNPs and production of a strong PA signal instead (Figure 9a). Cytotoxicity studies demonstrated no toxicity of α-CD/UCNPs contrast agents. Following in vitro imaging (Figure 9b), the first PAI in a living mouse was conducted using 980 nm excitation laser. These results demonstrated the ability of α-CD/UCNPs to be an efficient PAI contrast agent for diagnostic purposes [49].

**Figure 9.** (**a**) High-resolution PA signal originated from upconversional NPs (UCNPs) in cyclohexane (black curve), distilled water (green curve), and α-CD/UCNPs (red curve) in water. The excitation was conducted using 980 nm nanosecond pulsed laser. (**b**) Photo-acoustic imaging (PAI) of tissuemimicking phantom containing chambers filled with α-CD/UCN in water (green arrow) and distilled water (white arrow) [49]. The images are reprinted with permission from the publisher [49]. **Figure 9.** (**a**) High-resolution PA signal originated from upconversional NPs (UCNPs) in cyclohexane (black curve), distilled water (green curve), and α-CD/UCNPs (red curve) in water. The excitation was conducted using 980 nm nanosecond pulsed laser. (**b**) Photo-acoustic imaging (PAI) of tissue-mimicking phantom containing chambers filled with α-CD/UCN in water (green arrow) and distilled water (white arrow) [49]. The images are reprinted with permission from the publisher [49].

demonstrated the ability to serve as a contrast agent for non-invasive diagnostic of PJI [48].

tumor-selective theranostic agent [47].

An effective PAI agent for imaging prosthetic joint infection (PJI) [48] was demonstrated by conjugation of β-CD to indocyanine green (ICG). The β-CD-ICG PAI agent was demonstrated in the

Yu et al. recently developed a CD-based PAI contrast agent sensitive to tumor environment [47]. Gold NPs (AuNP) were modified initially with pyridine-2-imine-terminated single stand DNA via gold-thiol bonds, and α-CDs were capped on the end of DNA through hydrophobic interaction with CD's cavity. The α-CD-AuNP agent produced no PA signal under neutral pH conditions, but upon entering the tumor, α-CDs separate from the DNA ends due to reduction in non-covalent forces. This study demonstrated that a developed α-CD-AuNP contrast agent can be successfully used as a

An effective PAI agent for imaging prosthetic joint infection (PJI) [48] was demonstrated by conjugation of β-CD to indocyanine green (ICG). The β-CD-ICG PAI agent was demonstrated in the mice model of PJI. Wang et al. found that conjugation of ICG to β-CD improves its PA signal generation. Although the PAI signal increase was not significant with β-CD-ICG contrast, it still demonstrated the ability to serve as a contrast agent for non-invasive diagnostic of PJI [48].

Yu et al. recently developed a CD-based PAI contrast agent sensitive to tumor environment [47]. Gold NPs (AuNP) were modified initially with pyridine-2-imine-terminated single stand DNA via gold-thiol bonds, and α-CDs were capped on the end of DNA through hydrophobic interaction with CD's cavity. The α-CD-AuNP agent produced no PA signal under neutral pH conditions, but upon entering the tumor, α-CDs separate from the DNA ends due to reduction in non-covalent forces. This study demonstrated that a developed α-CD-AuNP contrast agent can be successfully used as a tumor-selective theranostic agent [47].
