2.1.2. In Vivo Imaging of the Host–Guest CD-Based Contrast Agents

The performance of some CD-based MRI imaging agents has been evaluated in vivo. Lahrech et al. demonstrated imaging of C6 glioma rats using a Gd-α-CD complex to quantify cerebral blood volume [62]. Although performance of Gd-α-CD contrast agents was significantly better compared to Gd-DOTA in terms of relaxation rates (r<sup>1</sup> <sup>=</sup> 7.3 mM−<sup>1</sup> s <sup>−</sup><sup>1</sup> at 9.4 T), the developed supramolecular agent did not accumulate in tumors and almost no enhancement was observed on T1-weighted images during the first hour after Gd-α-CD injection. On the contrary, the CBV fraction was successfully measured using rapid steady state T<sup>1</sup> method and Gd-α-CD contrast agent.

More advanced CD-based contrast agents were demonstrated by Sun et al. who created a supramolecular complex between bridged bis(permethyl-β-cyclodextrin)s with Mn-porphyrin bearing polyethylene glycol side chains (Mn-TPP) [63]. Mice injected with this supramolecular polymer demonstrated strong contrast observed in the blood, kidneys, and bladder (Figure 2) [63]. A supramolecular polymer built using the non-covalent interaction between Mn(II)-TPP and bridged tris(permethyl-β-CD)s resulted in a longitudinal relaxivity only 7% higher compared to previously developed Mn(II)-containing linear polymer [63,64].

Work by Feng et al. conducted on CD-based NPs as MRI contrast agents demonstrated neodymium doped NaHoF<sup>4</sup> NPs as T<sup>2</sup> imaging agents cultured with human mesenchymal stem cells injected into the brain hemisphere of nude mice. This combination of CD-based contrast agents and stem cells [23] supports the idea of using stem cells as an MRI contrast agent carrier. Furthermore, due to the high relaxivity of the developed probe (r<sup>2</sup> <sup>=</sup> 143.7 mM−<sup>1</sup> s <sup>−</sup><sup>1</sup> at 11.7 T) [23], use of the developed contrast agent will be beneficial for ultrahigh field MRI imaging, since the transverse relaxivity increases highly with the magnetic field strength [23,65,66].

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**Figure 2.** (**a**) Molecular structure of Mn(II)-TPP and bridged bis(permethyl-β-CD)s polymer. (**b**) Representative 2D coronal T1-weighted MR images of the mice at 2, 5, 10, 20, and 25 min after intravenous injection of Mn(II)-TPP/bridged β-CD magnetic resonance imaging (MRI) contrast agents at 0.03 mmol of Mn/kg [63]. The images are reprinted with permission from publisher [63]. **Figure 2.** (**a**) Molecular structure of Mn(II)-TPP and bridged bis(permethyl-β-CD)s polymer. (**b**) Representative 2D coronal T1-weighted MR images of the mice at 2, 5, 10, 20, and 25 min after intravenous injection of Mn(II)-TPP/bridged β-CD magnetic resonance imaging (MRI) contrast agents at 0.03 mmol of Mn/kg [63]. The images are reprinted with permission from publisher [63].

#### Work by Feng et al. conducted on CD-based NPs as MRI contrast agents demonstrated 2.1.3. In Vivo Tumor Imaging

neodymium doped NaHoF4 NPs as T2 imaging agents cultured with human mesenchymal stem cells injected into the brain hemisphere of nude mice. This combination of CD-based contrast agents and stem cells [23] supports the idea of using stem cells as an MRI contrast agent carrier. Furthermore, due to the high relaxivity of the developed probe (r2 = 143.7 mM−1s−1 at 11.7 T) [23], use of the developed contrast agent will be beneficial for ultrahigh field MRI imaging, since the transverse relaxivity increases highly with the magnetic field strength [23,65,66]. 2.1.3. In Vivo Tumor Imaging Imaging of cancer is one of the hot topics in modern medical imaging field. Despite the numerous developed contrast agents discussed above, only Zhou et al. used multiple β-CDs attached to a polyhedral oligomeric silsesquioxane nano globule at a targeted nano globular contrast agent from host–guest assembly for magnetic resonance cancer molecular imaging [67]. The host–guest contrast agent bonds to αvβ<sup>3</sup> integrinin 4T1 malignant breast tumor through cyclic RGDfK peptide and gives greater contrast enhancement, due to the αvβ<sup>3</sup> that is overexpressed in tumors (Figure 3a–c). This designed contrast agent produced superior contrast and signal enhancement compared to the clinically used Gd-based ProHance and non-targeted control cRAD-POSS-bCD-(DOTA-Gd)-Cy5

Imaging of cancer is one of the hot topics in modern medical imaging field. Despite the numerous developed contrast agents discussed above, only Zhou et al. used multiple β-CDs attached to a polyhedral oligomeric silsesquioxane nano globule at a targeted nano globular contrast agent

contrast agent. Molecular structure of cRGD-POSS-βCD-(DOTA-Gd)-Cy5 imaging agent is shown on Figure 3d. clinically used Gd-based ProHance and non-targeted control cRAD-POSS-bCD-(DOTA-Gd)-Cy5 contrast agent. Molecular structure of cRGD-POSS-βCD-(DOTA-Gd)-Cy5 imaging agent is shown on Figure 3d.

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from host–guest assembly for magnetic resonance cancer molecular imaging [67]. The host–guest contrast agent bonds to αvβ3 integrinin 4T1 malignant breast tumor through cyclic RGDfK peptide

**Figure 3.** Magnetic resonance molecular imaging with cRGD-POSS-βCD-(DOTA-Gd)-Cy5 in mice bearing 4T1-Luc2-CFP tumor xenografts. The representative 2D axial fat-suppressed T1-weighted spin-echo MRI images before and at 5, 15, and 30 min post-injection of ProHance (**a**), cRAD-POSSbCD-(DOTA-Gd)-Cy5 (**b**), and cRGD-POSS-bCD-(DOTA-Gd)-Cy5 (**c**) at 0.1 mmol-Gd/kg. The injection of cRGD-POSS-βCD-(DOTA-Gd)-Cy5 creates superior signal enhancement in tumor region. The images are reprinted with permission from publisher [67]. (**d**) Molecular structure of the developed cRGD-POSS-βCD-(DOTA-Gd)-Cy5 contrast agent. **Figure 3.** Magnetic resonance molecular imaging with cRGD-POSS-βCD-(DOTA-Gd)-Cy5 in mice bearing 4T1-Luc2-CFP tumor xenografts. The representative 2D axial fat-suppressed T1-weighted spin-echo MRI images before and at 5, 15, and 30 min post-injection of ProHance (**a**), cRAD-POSS-bCD-(DOTA-Gd)-Cy5 (**b**), and cRGD-POSS-bCD-(DOTA-Gd)-Cy5 (**c**) at 0.1 mmol-Gd/kg. The injection of cRGD-POSS-βCD-(DOTA-Gd)-Cy5 creates superior signal enhancement in tumor region. The images are reprinted with permission from publisher [67]. (**d**) Molecular structure of the developed cRGD-POSS-βCD-(DOTA-Gd)-Cy5 contrast agent.

#### *2.2. Direct Labeling of the CD Molecules 2.2. Direct Labeling of the CD Molecules*

Another approach of synthesis of CD-based contrast agents for MRI imaging purposes is to conjugate metal–organic Gd(III)-containing complexes to the CD molecule through covalent bonds. We refer to this method as direct labeling of CD molecules. The key advantage of this approach is the Another approach of synthesis of CD-based contrast agents for MRI imaging purposes is to conjugate metal–organic Gd(III)-containing complexes to the CD molecule through covalent bonds. We refer to this method as direct labeling of CD molecules. The key advantage of this approach is the availability of CD cavity for host–guest interaction with other molecules that can be effectively used for drug delivery study and imaging of molecular interactions.

Synthetically, modifying CDs in order to meet the criteria of an ideal contrast agent can be summarized by three key components: having a point of functionalization to attach the chelating group, designing a rigid linker in order to slow local movements, and lastly, conjugation of the macrocyclic complex in order to encapsulate multiple lanthanide chelates—thus, overall enhancing MRI signal, while improving overall stability and relaxivity profiles [35,54–56,68]. Synthetically, modifying CDs in order to meet the criteria of an ideal contrast agent can be summarized by three key components: having a point of functionalization to attach the chelating group, designing a rigid linker in order to slow local movements, and lastly, conjugation of the macrocyclic complex in order to encapsulate multiple lanthanide chelates—thus, overall enhancing MRI signal, while improving overall stability and relaxivity profiles [35,54–56,68]. Various synthetic approaches have been explored, which demonstrates CD's ability to be

for drug delivery study and imaging of molecular interactions.

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Various synthetic approaches have been explored, which demonstrates CD's ability to be functionalized and conjugated in a robust fashion dependent on the desired application(s) [35,54–56,68]. Bryson et al. synthesized a monodisperse β-CD Click cluster containing seven paramagnetic chelates encompassing two water exchange sites [35]. Using Click chemistry, an alkyne-functionalized dendron was reacted with the per-azido-β-CD to yield the desired product (Figure 4). Using similar methods, Champagne et al. recently reported the synthesis of a different β-CD MRI probe containing seven iminodiacetate arms connected at the C6-position of β-CD by a triazole-based linker following a copper(I)-mediated 1,3-dipolar cycloaddition [54]. In addition, CDs can be differentially conjugated to produce multifunctional probes. Kotková et al. synthesized a novel bimodal fluorescence/MRI probe using a β-CD scaffold [55]. β-CD was labeled first using fluorescein isothiocyanate (FITC) and subsequentially with an isothiocyanate derivative containing a DOTA-based ligand [55]. The rigidity of the linker between the CD and the Gd-containing ligand plays an important role in increasing TR, thus enhancing the overall MRI signal [35,55]. Additionally, multifunctional NPs have been modified using an asymmetrically functionalized β-CD-based star copolymer by conjugating β-CD using doxorubicin (DOX), folic acid (FA), and DOTA-Gd moieties [54]. Similar to the work of Bryson et al. [35] and Champagne et al. [54], the key conjugation method used was azide-alkyne Huigsen cycloaddition, creating rigid triazole linkers. functionalized and conjugated in a robust fashion dependent on the desired application(s) [35,54– 56,68]. Bryson et al. synthesized a monodisperse β-CD Click cluster containing seven paramagnetic chelates encompassing two water exchange sites [35]. Using Click chemistry, an alkynefunctionalized dendron was reacted with the per-azido-β-CD to yield the desired product (Figure 4). Using similar methods, Champagne et al. recently reported the synthesis of a different β-CD MRI probe containing seven iminodiacetate arms connected at the C6-position of β-CD by a triazole-based linker following a copper(I)-mediated 1,3-dipolar cycloaddition [54]. In addition, CDs can be differentially conjugated to produce multifunctional probes. Kotková et al. synthesized a novel bimodal fluorescence/MRI probe using a β-CD scaffold [55]. β-CD was labeled first using fluorescein isothiocyanate (FITC) and subsequentially with an isothiocyanate derivative containing a DOTAbased ligand [55]. The rigidity of the linker between the CD and the Gd-containing ligand plays an important role in increasing TR, thus enhancing the overall MRI signal [35,55]. Additionally, multifunctional NPs have been modified using an asymmetrically functionalized β-CD-based star copolymer by conjugating β-CD using doxorubicin (DOX), folic acid (FA), and DOTA-Gd moieties [54]. Similar to the work of Bryson et al. [35] and Champagne et al. [54], the key conjugation method used was azide-alkyne Huigsen cycloaddition, creating rigid triazole linkers.

**Figure 4.** Multivalent β-CD "Click cluster", containing seven paramagnetic chelating groups, each with two water exchange sites linked via triazole-based linkers. The β-CD "Click cluster" was synthesized from per-azido-β-CD precursor and conjugated using the well-established Huigsen cycloaddition reaction. Figure adapted from Bryson et al. with permission from publisher [35]. **Figure 4.** Multivalent β-CD "Click cluster", containing seven paramagnetic chelating groups, each with two water exchange sites linked via triazole-based linkers. The β-CD "Click cluster" was synthesized from per-azido-β-CD precursor and conjugated using the well-established Huigsen cycloaddition reaction. Figure adapted from Bryson et al. with permission from publisher [35].

#### 2.2.1. In Vitro Development 2.2.1. In Vitro Development

Skinner et al. demonstrated the first labeling of CD macrocycle with a Gd(III) chelate albeit the CD cavity was still used for non-covalent binding to another Gd(III) chelate in order to increase the proton relaxivity. [69]. Relaxivity of this complex increased when it was bound noncovalently to Skinner et al. demonstrated the first labeling of CD macrocycle with a Gd(III) chelate albeit the CD cavity was still used for non-covalent binding to another Gd(III) chelate in order to increase the proton relaxivity. [69]. Relaxivity of this complex increased when it was bound noncovalently to another gadolinium complex with the addition of two phenyl moieties.

another gadolinium complex with the addition of two phenyl moieties. Bryson et al. created a contrast agent with a ten-fold increase in relaxivity at 9.4 T compared to clinically available Magnevist by labeling of per-azido-β-cyclodextrin core with seven

diethylenetriaminetetraacetic acid (DTTA) Gd(III) chelates [35]. This development can be attributed to the increased Gd(III) ions per molecule and further increase in relaxivity due to conjugation to the macrocycle. In addition to the high relaxivity, unoccupied cavity of β-CD makes the developed contrast agent an excellent host scaffold to functionalize through noncovalent assembly with biological receptor-specific targets.

Bimodal MRI-fluorescence probes were demonstrated by Kotkova et al. [55] who combined a DOTA-based ligand with fluorescein functionality to simultaneously obtain fluorescence and MR images. Although the developed CD-based agent was studied in vitro, the benefit of this scaffold for MRI visualization under in vivo conditions was assumed due to its low cytotoxicity and high cell uptake. Fredy et al. developed cyclodextrin polyrotaxanes as a highly modular platform for an imaging agent [70]. Selectively functionalized cyclodextrins with a Gd(III) complex or BODIPY fluorescent tag were put on to a polyammonium chain to form polyrotaxanes. From this, polyrotaxanes could be assembled with fluorescent CDs and CDs with dia- or paramagnetic lanthanide complexes. Each threaded cyclodextrin was molecularly defined, which is an advantage over statistical post-functionalization of CD-polyrotaxanes. In vitro studies demonstrated that the Gd-bearing polyrotaxanes have relaxivities that are five times higher than Gd-DOTA, which makes them effective contrast agents for MRI applications [70].

NPs fabricated using biological macromolecules have been demonstrated by both Liu et al. [68] and Su et al. [71]. Liu designed pH disintegrable β-CD-based micellar NPs, while Su reported star-like dextran wrapped superparamagnetic iron oxide NPs. Both groups reported dual effects: an imaging contrast agent and cytotoxicity to HeLa cells at high concentrations, making these molecules both imaging probes and potential chemotherapeutics agents. Later, the synthesis of the contrast agent that affects the spin-spin (T2) relaxation was suggested by conjugating β-CD to magnetic NPs [72].
