*2.4.3. Mannose and fucose-appended α-CDEs as Kupffer cell (KC)-selective pDNA and siRNA carriers*

KCs are reticuloendothelial cells that reside within the lumen of the liver sinusoid and adhere to endothelial cells that form blood-vessel walls. These non-parenchymal cells represent ~15% of the total liver cells in the human body [65] and are the first macrophages that come into contact with bacteria and bacterial toxins derived from the gastrointestinal tract [66]. KCs also play a critical role in removing harmful materials circulating in the blood. Moreover, KCs are considered an essential part of innate immunity and play an important role in the rapid response to threatening stimuli. They are also involved in the pathogenesis of different liver diseases, including viral hepatitis, alcoholic liver disease, non-alcoholic fatty liver, development of liver fibrosis, and portal hypertension [67]. Importantly, KCs express ManR and FucR; therefore, both fucose and mannose can be used as targeting ligands on KCs used for nucleic acid delivery [68].

Arima et al. prepared mannose-appended α-CDEs (Man-α-CDE, G2) [39] to develop ManRtargeted non-viral carriers by attaching mannose residues to primary amino acid residues of α-CDE (G2) using an α-D-mannopyranosylphenyl isothiocyanate spacer. Of the various Manα-CDEs (G2) with degrees of substitution of mannose (DSMs) of 1.1, 3.3, 4.9, and 8.3, Man-α-CDEs (G2, DSM3.3 and DSM4.9) showed higher gene-transfer activity as compared with that of dendrimer (G2) and α-CDE (G2) in NR8383 cells, a rat lung macrophage cell line, with no cytotoxicity observed up to charge ratio of 200 (carrier/pDNA). However, Man-α-CDE (G2) also showed high gene-transfer activity in A549 cells [ManR (−)], suggesting its low selectivity for ManR, possibly due to the rigidity of the spacer.

More recently, Arima et al. prepared a new Man-α-CDE with a α-D-mannopyranosylprop ylthiopropionylated α-CDE (G3) spacer [Man-S-α-CDEs (G3)], which was longer and more flexible than that in Man-α-CDE [69, 70]. In this study, nuclear factor (NF)-κB was targeted due to its important role in the inflammatory response, and because it is found in almost all animal cell types. Therefore, to suppress NF-κB activation, NF-κB siRNA and NF-κB-decoy DNA were employed, with both strategies potentially attractive for the treatment of cytokinerelated liver diseases, such as fulminant hepatitis. Of the various Man-S-α-CDEs with different DSMs, the NF-κB p65-specific siRNA (sip65) complex with Man-S-α-CDE (G3, DSM4) showed significantly lower *NF-*κ*B p65* mRNA levels and nitric oxide levels in lipopolysaccharide (LPS)-stimulated NR8383 cells following ManR-mediated endocytosis (**Figure 3**). Intravenous administration of the Man-S-α-CDE (G3, DSM4)/sip65 complex increased the survival rate of the LPS-induced fulminant hepatitis mouse model via significant *in vivo* RNAi activity. These results suggested that Man-S-α-CDE (G3, DSM4) represented a potential novel KC-selective siRNA carrier.

Several reports demonstrated that NF-κB-decoy complexes harboring a liposome-functionalized fucose moiety showed higher gene-transfer efficiency as compared with mannoseappended liposomes specific for KCs [71, 72]. Therefore, Akao et al. prepared thioalkylated fucose-appended α-CDEs [Fuc-S-α-CDE (G2)] and assessed their potential as KC-selective carriers of decoy DNA (**Figure 4**) [73]. The NF-κB-decoy in complex with Fuc-S-α-CDE (G2) with an average degree of substitution of fucose (DSFuc) of two suppressed the production of

**Figure 2.** PEG-LαC (G3) as a targeting carrier for siTTR delivery to hepatocytes.

Recently, Hayashi et al. prepared a PEGylated Lac-α-CDE [PEG-LαC (G3)] to improve *in vivo* gene-transfer activity by enhancing complex stability, as well as prolonging the half-life in circulation [62]. Of the various PEG-LαCs (G3), those with degrees of substitution of the PEG moiety (DSPs) of 2.1 [PEG-LαC (G3, DSP2.1] showed higher luciferase gene-transfer activity than other PEG-LαCs (G3, DSP4.0 and DSP6.2) in HepG2 cells along with negligible cytotoxicity up to a charge ratio of 50. Additionally, its gene-transfer activity decreased in the presence of asialofetuin, whereas it retained significantly higher gene-transfer activity, even in the presence of 50% serum. Additionally, PEG-LαC (G3, DSP2.1) showed selective genetransfer activity into hepatic parenchymal cells rather than hepatic non-parenchymal cells *in vivo*. Furthermore, blood-chemistry values, such as CRE, BUN, AST, ALT, and LDH concentrations, following administration of the PEG-LαC (G3, DSP2.1)/pDNA complex system were almost equivalent with those in controls, suggesting that PEG-LαC (G3, DSP2.1) showed

The potential of PEG-LαC (G3) as a siRNA carrier was also evaluated (unpublished data), with an siRNA against transthyretin (TTR) mRNA (siTTR) used to treat TTR-related familial amyloidotic polyneuropathy (TTR-FAP). The results indicated that the PEG-LαC (G3)/siTTR complex significantly reduced TTR mRNA expression in the liver as compared with the Lacα-CDE (G3)/siTTR complex, suggesting the potential use of PEG-LαC (G3) as a hepatocyte-

potential as a hepatocyte-selective gene carrier both *in vitro* and *in vivo*.

selective siRNA carrier (**Figure 2**).

248 Cyclodextrin - A Versatile Ingredient

nitric oxide, as well as tumor necrosis factor-α (TNF-α) expression, in LPS-simulated NR8383 cells through FucR-mediated cellular uptake and successful endosomal escape. This complex also improved survival rates following intravenous injection into a fulminant hepatitis mouse model. Moreover, Fuc-S-α-CDE (G2, DSFuc2)/NF-κB decoy complexes showed marked accumulation in the liver relative to that observed in other organs. Furthermore, serum ALT and AST levels, as well as TNF-α levels, significantly decreased after intravenous administration of the complex in mice with fulminant hepatitis. These results suggested the potential of the Fuc-S-α-CDE (G2)/NF-κB decoy complex as an oligonucleotide therapy for fulminant hepatitis. There are many other receptors available for KC-specific drug targeting, including galactose receptors, scavenger receptors, CD36, lol-density lipoprotein receptor, and complement receptors [74]; therefore, future studies on the utility of fucose in this context should focus on its efficacy with different ligands.

*2.4.4. Folate PEG-appended α-CDEs as cancer-cell-selective pDNA and siRNA carriers*

**Figure 4.** Fuc-S-α-CDE (G2) as targeting decoy DNA carrier to KCs.

To achieve maximum effective therapeutic effects against cancer using siRNAs, the design of tumor-selective delivery systems is extremely crucial. Folic acid has often been used as a tumor-specific ligand [75, 76], because it is relatively affordable as compared with other cancer-targeting ligands and is capable of high-affinity interactions with the FR-α receptor

Promising Use of Cyclodextrin-Based Non-Viral Vectors for Gene and Oligonucleotide Drugs

http://dx.doi.org/10.5772/intechopen.74614

251

**Figure 3.** Man-S-α-CDE (G3) as a KC-specific targeting carrier via ManR.

#### *2.4.4. Folate PEG-appended α-CDEs as cancer-cell-selective pDNA and siRNA carriers*

nitric oxide, as well as tumor necrosis factor-α (TNF-α) expression, in LPS-simulated NR8383 cells through FucR-mediated cellular uptake and successful endosomal escape. This complex also improved survival rates following intravenous injection into a fulminant hepatitis mouse model. Moreover, Fuc-S-α-CDE (G2, DSFuc2)/NF-κB decoy complexes showed marked accumulation in the liver relative to that observed in other organs. Furthermore, serum ALT and AST levels, as well as TNF-α levels, significantly decreased after intravenous administration of the complex in mice with fulminant hepatitis. These results suggested the potential of the Fuc-S-α-CDE (G2)/NF-κB decoy complex as an oligonucleotide therapy for fulminant hepatitis. There are many other receptors available for KC-specific drug targeting, including galactose receptors, scavenger receptors, CD36, lol-density lipoprotein receptor, and complement receptors [74]; therefore, future studies on the utility of fucose in this context should focus on

its efficacy with different ligands.

250 Cyclodextrin - A Versatile Ingredient

**Figure 3.** Man-S-α-CDE (G3) as a KC-specific targeting carrier via ManR.

To achieve maximum effective therapeutic effects against cancer using siRNAs, the design of tumor-selective delivery systems is extremely crucial. Folic acid has often been used as a tumor-specific ligand [75, 76], because it is relatively affordable as compared with other cancer-targeting ligands and is capable of high-affinity interactions with the FR-α receptor

**Figure 4.** Fuc-S-α-CDE (G2) as targeting decoy DNA carrier to KCs.

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 ideal candidate cancer-cell-selective ligand.

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

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

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