**2.5. 6-***O***-α-(4-***O***-α-D-glucuronyl)-D-glucosyl (GUG)-β-CDE as a pDNA and siRNA carrier**

Arima et al. clarified the importance of a spacer between the dendrimers and the targeting ligands for providing cell-specific pDNA delivery [78, 80]. However, the effect of a spacer between the CyD and the dendrimer on the gene-transfer activity of the CDE remained unknown. Consequently, a new CDE was prepared (GUG-β-CDE) utilizing a glucuronyl-glucosyl group as a spacer between the CyD and the dendrimer. Additionally, GUG-β-CyD has many advantages over the parent β-CyD, including higher water solubility, lower hemolytic activity, and increased bioadaptability [81]. Moreover, it contains a carboxyl group capable of interacting with primary amino groups present in dendrimers. Of the various GUG-β-CDEs (G2) having different DS values, GUG-β-CDE (G2, DS1.8) showed higher gene-transfer activity *in vitro* as compared with other GUG-β-CDEs (DS1.2, DS2.5, and DS4.5) [82]. Additionally, GUG-β-CDE (G2, DS1.8) showed higher gene-transfer activity relative to that of α-CDE (G2, DS1.2) and β-CDE

(G2, DS1.3) in A549 cells and RAW264.7 cells, respectively, possibly due to the pDNA complex exhibiting an increased ability to escape endosomes and a high degree of nuclear localization [39, 83, 84]. Moreover, *in vivo* GUG-β-CDE (G2, DS1.8) gene-transfer activity was much higher than that of α-CDE (G2, DS1.2) or β-CDE (G2, DS1.3) in kidney at 12 h after intravenous injection of the complexes in mice [85]. Therefore, GUG-β-CDE (DS1.8) might have potential as a pDNA carrier for gene-transfer targeting the kidney. Furthermore, no cytotoxicity was observed in A549 cells or RAW264.7 cells up to a charge ratio of 200 (carrier/pDNA). The hemolytic activity of GUGβ-CDE (G2, DS1.8) in rabbit red blood cells was also substantially lower than that associated with the dendrimer, and negligible changes in the blood-chemistry data were observed 12 h after

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**Figure 5.** Fol-PαC (G4) as a targeting-siRNA carrier to folate-expressing tumor cells.

Promising Use of Cyclodextrin-Based Non-Viral Vectors for Gene and Oligonucleotide Drugs http://dx.doi.org/10.5772/intechopen.74614 253

**Figure 5.** Fol-PαC (G4) as a targeting-siRNA carrier to folate-expressing tumor cells.

expressed on the surface of many cancer cells (*kd* > 10−9–10−10 M). FR-α is highly expressed in several tumor cells, including those associated with lung, ovary, breast, kidney, and brain cancers, and is negligibly expressed in normal tissues. Additionally, as the cancer progresses in stage, the FR-α expression increases substantially. Therefore, folic acid is considered an

Arima et al. prepared a folic acid-appended α-CDE (G3) with a PEG spacer [Fol-PαC (G3)] to fabricate a cancer-selective gene and siRNA carrier. Fol-PαC (G3) showed selective FR-αoverexpressing tumor-cell gene-transfer activity [77]. Specifically, Fol-PαC (G3) with an average degree of substitution of folate (DSF) of five showed significantly higher gene-transfer activity as compared with that of α-CDE (G3) in KB cells [FR-α (+)], but not in A549 [FR-α (−)] cells along with negligible cytotoxicity. Moreover, Fol-PαC (G3, DSF5) showed higher gene-

The potential of Fol-PαC (G3) for delivery of siRNA to FR-α-overexpressing cancer cells was evaluated [78], showing that Fol-PαC (G3, DSF4) exhibited high siRNA-transfer activity in KB cells [FR-α (+)] in the absence of cytotoxicity up to a charge ratio of 100 (carrier/siRNA). Notably, the Fol-PαC (G3, DSF4)/siRNA complex showed significant RNAi activity following

Ohyama et al. then prepared Fol-PαCs using a higher-generation dendrimer (G4) and evaluated their potential as tumor-targeting siRNA carriers *in vitro* and *in vivo* [79]. The Fol-PαC (G4, DSF2)/siRNA complex showed prominent RNAi activity based on adequate physicochemical properties, FR-α-mediated endocytosis, efficient endosomal escape, and siRNA delivery to the cytoplasm along with negligible cytotoxicity (**Figure 5**). Most importantly, Fol-PαC (G4, DSF2) showed improved siRNA-specific blood-circulating ability, serum stability, and *in vivo* RNAi activity as compared with those observed with Fol-PαC (G3). Additionally, Fol-PαC (G4, DSF2) in complex with siRNA against Polo-like kinase 1 (siPLK1) suppressed tumor growth as compared with that observed using a control siRNA complex in mice bearing colon-26 tumor cells. These results suggested that Fol-PαC (G4) represented a potential

transfer activity than α-CDE (G3) after intratumoral injection in mice bearing tumors.

intratumoral injection; however, this was not the result of its dissociation in blood.

**2.5. 6-***O***-α-(4-***O***-α-D-glucuronyl)-D-glucosyl (GUG)-β-CDE as a pDNA and siRNA** 

Arima et al. clarified the importance of a spacer between the dendrimers and the targeting ligands for providing cell-specific pDNA delivery [78, 80]. However, the effect of a spacer between the CyD and the dendrimer on the gene-transfer activity of the CDE remained unknown. Consequently, a new CDE was prepared (GUG-β-CDE) utilizing a glucuronyl-glucosyl group as a spacer between the CyD and the dendrimer. Additionally, GUG-β-CyD has many advantages over the parent β-CyD, including higher water solubility, lower hemolytic activity, and increased bioadaptability [81]. Moreover, it contains a carboxyl group capable of interacting with primary amino groups present in dendrimers. Of the various GUG-β-CDEs (G2) having different DS values, GUG-β-CDE (G2, DS1.8) showed higher gene-transfer activity *in vitro* as compared with other GUG-β-CDEs (DS1.2, DS2.5, and DS4.5) [82]. Additionally, GUG-β-CDE (G2, DS1.8) showed higher gene-transfer activity relative to that of α-CDE (G2, DS1.2) and β-CDE

novel tumor-targeting siRNA carrier *in vitro* and *in vivo*.

**carrier**

ideal candidate cancer-cell-selective ligand.

252 Cyclodextrin - A Versatile Ingredient

(G2, DS1.3) in A549 cells and RAW264.7 cells, respectively, possibly due to the pDNA complex exhibiting an increased ability to escape endosomes and a high degree of nuclear localization [39, 83, 84]. Moreover, *in vivo* GUG-β-CDE (G2, DS1.8) gene-transfer activity was much higher than that of α-CDE (G2, DS1.2) or β-CDE (G2, DS1.3) in kidney at 12 h after intravenous injection of the complexes in mice [85]. Therefore, GUG-β-CDE (DS1.8) might have potential as a pDNA carrier for gene-transfer targeting the kidney. Furthermore, no cytotoxicity was observed in A549 cells or RAW264.7 cells up to a charge ratio of 200 (carrier/pDNA). The hemolytic activity of GUGβ-CDE (G2, DS1.8) in rabbit red blood cells was also substantially lower than that associated with the dendrimer, and negligible changes in the blood-chemistry data were observed 12 h after intravenous administration of the GUG-β-CDE (G2, DS1.8)/pDNA complex in mice. These results strongly suggested that this complex showed a good safety profile *in vivo* and *in vitro*, and that it might constitute an adequate carrier for gene therapy targeting kidney diseases, such as polycystic kidney disease, Alport syndrome, renal cancers, glomerulonephritis, and renal fibrosis.

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Additionally, Anno et al. evaluated the potential of GUG-β-CDE (G2) as a siRNA carrier. GUG-β-CDE (G2, DS1.8) in complex with siTTR showed high RNAi activity with no cytotoxicity in HepG2 cells. Moreover, *TTR* mRNA-expression levels were reduced after intravenous administration of the complex to BALB/c mice, with only minor changes in blood-chemistry parameters, suggesting the potential of GUG-β-CDE (G2, DS1.8) as a siRNA carrier for the treatment of TTR-FAP [86].

Moreover, Ahmed et al. prepared a GUG-β-CDE using a higher-generation dendrimer (G3) [87]. Various GUG-β-CDEs (G3, DS1.6, DS3.0, DS3.7, DS5.0, and DS8.6) were prepared, with the GUG-β-CDE (G3, DS3.7)/siRNA complex showing the highest RNAi activity in KB cells transiently expressing the luciferase gene and colon 26-luc cells stably expressing the luciferase gene. Moreover, the GUG-β-CDE (G3, DS3.7)/FITC-siRNA complex showed the highest cellular uptake along with negligible cytotoxicity at a charge ratio of 20 (carrier/siRNA). Additionally, cellular uptake of the GUG-β-CDE (G3, DS3.7)/FITC-siRNA complex was significantly higher than that of the α-CDE (G3, DS2.4)/FITC-siRNA complex, suggesting GUGβ-CDE (G3, DS3.7) as a potential effective siRNA carrier. Currently, folate-PEG-appended GUG-β-CDEs (G3) are in development as a cancer-selective GUG-β-CDE (G3) variant.

## **2.6. Conclusion**

In this review, we described various CDEs used as gene and oligonucleotide carriers. These multifunctional CDEs showed great potential as carriers for DNA and nucleic acid drugs. The advantages of these CDEs included (1) low cytotoxicity; (2) facile modification of various targeting ligands and polymers, such as PEG; and (3) enhanced endosomal-escape ability via the synergistic action of the proton-sponge effect in the dendrimer and the interaction of CyD with membrane lipids. Therefore, these CyD-based carriers have the potential for utilization as multifunctional carriers for pDNA, siRNA, decoy DNA, and shRNA.
