**2. Polyamidoamine (PAMAM) dendrimer conjugates with α-CyDs (α-CDEs) as nucleic acid carriers**

#### **2.1. α-CDEs as plasmid (p) DNA carriers**

developing modified dendrimers possessing high levels of safety along with low numbers of

**Figure 1.** PAMAM dendrimer conjugates with various CyD (CDEs) and targeting ligands. GUG-β-CyD, glucuronylglucosylβ-CyD; Man-α-CDE, mannosylated-CDE; Gal-α-CDE, galactosylated-CDE; Fol-PαC, folate-PEG-appended α-CDE; Man-S-α-CDE, mannosyloxypropylthioalkylated-α-CDE; Fuc-S-α-CDE, fucosyloxypropylthioalkylated-α-CDE; Lacα-CDE, lactosylated-CDE; PEG-LαC, PEGylated Lac-α-CDE; Lac-PαC, lactose-PEG-appended α-CDE; PAMAM,

The potential use of cyclodextrins (CyDs) as carriers of nucleic acids based of their direct interaction would not be expected, given that they exhibit very weak interactions with nucleic acids [30]. Therefore, combining CyDs with cell-penetrating nucleic acid carriers (cationic polymers) or modifying the CyD structure was necessary for their internalization. Various methods have been adopted to enhance the interactions between CyD polymers and conjugates with nucleic

generations due to their extremely low cytotoxicity [27–29].

polyamidoamine.

242 Cyclodextrin - A Versatile Ingredient

Arima et al. prepared a variety of CyD conjugates with PAMAM dendrimers (CDE) and utilized them as gene and nucleic acid drug carriers. Originally, Arima et al. prepared dendrimer [generation 2 (G2)] conjugates with α-, β-, and γ-CyDs and named them α-, β-, and γ-CDEs (G2), respectively [36]. Among these CDEs, α-CDE (G2) exhibited the most prominent genetransfer activity, showing 100-fold higher luciferase gene-transfer activity as compared with that of dendrimer (G2) alone or its physical mixture with α-CyD in NIH3T3 cells, a mouse embryo fibroblast cell line, and RAW264.7 cells, a mouse macrophage-like cell line. This was attributed not only to increased levels of cellular association, but also to the augmented endosomal-escape ability of the pDNA complex due to the synergy of both the proton-sponge effect and the ability of α-CyD to disrupt the endosomal membrane. Afterward, Kihara et al. examined the optimal dendrimer generation (G2, G3, or G4) and degree of substitution (DS) for α-CyD in the α-CDE molecule [37]. Consequently, α-CDE (G3, DS2) showed the highest transfection efficiency along with low cytotoxicity, which was superior to that of TransFact and Lipofectin when tested in NIH3T3 cells. Furthermore, to elucidate the reason behind the superior gene-transfer activity of α-CDE (G3, DS2), Arima et al. investigated the cellular uptake, intracellular distribution, and the physicochemical properties of pDNA complexes involving both α-CDE (G3, DS2) and the dendrimer (G3). The particle sizes, as well as the ζ-potential values, were nearly the same for both complexes. Furthermore, the enhanced gene-transfer activity could not be explained based on cellular uptake, as the values of the complexes with α-CDEs (G2, G3, and G4) were equivalent to those observed with their parent dendrimers, suggesting that factors other than cellular uptake or the physicochemical properties of the α-CDE (G3, DS2)/pDNA complexes might be strongly associated with improving gene-transfer activity. To elucidate the mechanism of cellular uptake, Arima et al. studied the effect of different endocytosis inhibitors on the cellular uptake of fluorescein isothiocyanatelabeled pDNA [(FITC)-pDNA] complexes with tetramethylrhodamine-5-(and 6)-isothiocyanate-labeled α-CDE [TRITC-α-CDE (G3)] transfection in A549 cells, ultimately using flow cytometry and confocal laser-scanning microscopy (CLSM) to observe the colocalization of TRITC-α-CDE (G3), FITC-endocytosis markers, and FITC-pDNA after transfection.

Consequently, after transfection of pDNA complexes, the complexes with TRITC-α-CDE (G3, DS2) colocalized with the endocytosis markers FITC-transferrin and FITC-cholera toxin B. Similarly, the gene-transfer activity of α-CDE (G3, DS2) was markedly lowered by the addition of clathrin-dependent endocytosis inhibitors (i.e., chlorpromazine and sucrose) and raft-dependent endocytosis inhibitors (i.e., nystatin and filipin), but not by amiloride, the macropinocytosis inhibitor. These results suggested that the main mechanism of α-CDE (G3, DS2) uptake involved clathrin- and raft-dependent endocytosis. To confirm the release of the complex from endosomes, we observed the intracellular distribution of the α-CDE (G3, DS2)/FITC-pDNA complex by CLSM, finding that the green fluorescence originating from FITC-pDNA in the case of the α-CDE (G3, DS2) complex was predominantly localized in the cytoplasm to a much greater degree than that of the dendrimer system, confirming the improved capability for endosomal disruption conferred by the synergy between α-CDE and the proton-sponge effect of the dendrimer.

similar to results observed with the α-CDE (G3, DS2)/pDNA complex, α-CDE (G3, DS2)/FITCsiRNA was delivered exclusively to the cytoplasm in NIH3T3-luc cells. Additionally, when this system was applied *in vivo* in mice bearing Colon-26-luc tumors, α-CDE (G3, DS2)/siGL3 (siRNA against pGL3 gene) showed potent RNAi activity against pGL3 expression after intravenous, as well as intratumoral, injection. Moreover, the siRNA complex neither triggered the immune response nor changed blood-chemistry data, indicating its safety. These results suggested the potential of α-CDE (G3, DS2) as a novel siRNA-carrier candidate for both *in* 

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To improve upon and prolong the duration of RNAi-mediated gene knockdown, vectorbased shRNA-expression systems were developed [42]. Upon shRNA transfection into mammalian cells, the insert containing the vector is transferred to the nucleus, integrated into the host genome, expressed, and quickly processed by Dicer-dependent cleavage and loaded into the RNA-induced silencing complex, which is then directed to the target mRNA, resulting in its degradation. A previous study demonstrated the potential of α-CDE (G3, DS2) as a novel carrier of pDNA expressing shRNA [43]. In this study, the authors used pDNA expressing shRNA against the pGL3 firefly luciferase gene (shGL3). Even at a low charge ratio, α-CDE (G3, DS2) capably formed a stable condensed complex with shGL3 and induced the conformational transition of shRNA in solution from B-form to the more compact C-form DNA. Furthermore, α-CDE (G3, DS2) markedly inhibited shGL3 degradation by DNase I, and the α-CDE (G3, DS2)/shGL3 complex showed the most potent RNAi activity at a charge ratio of 20 along with negligible cytotoxicity and without off-target effects in A549 cells, a human alveolar adenocarcinoma cell line, while also transiently expressing the luciferase gene in NIH3T3-luc cells. Moreover, the addition of sufficient amounts of siGL3 along with α-CDE (G3, DS2)/shGL3 dramatically enhanced the RNAi activity, which was ascribed to the stabilizing effect of α-CDE (G3, DS2) against DNase I degradation of the shRNA accompanied by negligible cytotoxicity. These results suggested that α-CDE (G3, DS2) possessed the potential

*vivo* and *in vitro* applications.

to be a novel shRNA carrier.

**2.4. Functionalized α-CDEs as cell-specific DNA and siRNA carriers**

In the early 1900s, the German Noble laureate Paul Ehrlich first introduced the concept of targeted drug delivery [44, 45]. At that time, he called it the "magic bullet", as it was able to deliver the drug specifically to microbes (such as bacteria) without harming the body. His continuous research eventually led to the development of the first effective drug against syphilis (Salvarsan). The primary aim of targeted drug-delivery systems is to increase the concentration of the medication in specific areas of the body relative to others, thereby improving its therapeutic index and reducing cytotoxicity. Various approaches have been adopted to target medications to the disease site [46, 47] with many compounds internalized inside of cells via receptor-mediated endocytosis. Receptor-mediated techniques use ligands attached to polyplexes to transfect cells with selected genes. Endocytosis is then mediated by various receptors, such as asialoglycoprotein receptor (ASGPR), mannose receptor (ManR), fucose

**2.3. α-CDE (G3) as shRNA carriers**

Moreover, *in vivo* studies of α-CDE (G3, DS2), as well as dendrimer complexes with pDNA, were evaluated after intravenous administration of 50 μg pDNA/mouse at a charge ratio of 10. After 12 h, organs were collected, and pDNA levels in various organs were determined. The results showed that α-CDE (G3, DS2) delivered pDNA more efficiently in the liver and kidney; however, the highest gene-expression levels were observed in the spleen. Bloodchemistry data related to α-CDE administration, including aspartate aminotransferase (AST), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), alanine aminotransferase (ALT), and creatinine (CRE) concentrations, showed minor changes as compared with those associated with the dendrimer [38]. These results suggested potential use of α-CDE (G3, DS2) as a safe and promising non-viral pDNA vector, although additional modifications of the α-CDE (G3, DS2) chemical structure were required to improve its nuclear translocation enable improved gene-expression results [39].

#### **2.2. α-CDE (G3) as siRNA carriers**

Assessment of the siRNA-carrier ability of α-CDE (G3, DS2) was performed by Tsutsumi et al. using a luciferase-reporter system [40, 41]. Their results showed significant reductions in the luciferase activity of the α-CDE (G3, DS2) system as compared with that observed in the control and accompanied by negligible cytotoxicity after transfection with the ternary complex pDNA/siRNA/α-CDE (G3, DS2). In this study, α-CDE (G3, DS2) showed superior transfection efficiency relative to that of the dendrimer and other commercially available transfection reagents. Additionally, they observed that the complex localized exclusively to the cytoplasm, where RNAi-related activity occurs, and as a result of the lack of a nuclear-translocation moiety in α-CDE (G3, DS2). Tsutsumi et al. also reported efficient knockdown of the firefly luciferase gene using a α-CDE (G3, DS2)/siRNA binary system accompanied by negligible cytotoxicity as compared with the use of siRNA complexes with commercially available transfection reagent (Lipofectamine 2000 and RNAiFect). Furthermore, the physicochemical properties, local irritation, cytotoxicity, interferon response, cellular uptake, and intracellular distribution of the siRNA complexes, as well as the RNAi activity associated with the α-CDE (G3, DS2)/siRNA complex, were evaluated on endogenous gene-expression in Colon-26-luc and NIH3T3-luc cells stably expressing the pGL3 luciferase gene. The results revealed potent RNAi activity against Lamin A/C and Fas expression along with minor cytotoxicity as compared with commercial transfection agents [40]. Additionally, siRNA complexed with α-CDE (G3, DS2) was protected from degradation by serum nucleases. Intriguingly and somewhat similar to results observed with the α-CDE (G3, DS2)/pDNA complex, α-CDE (G3, DS2)/FITCsiRNA was delivered exclusively to the cytoplasm in NIH3T3-luc cells. Additionally, when this system was applied *in vivo* in mice bearing Colon-26-luc tumors, α-CDE (G3, DS2)/siGL3 (siRNA against pGL3 gene) showed potent RNAi activity against pGL3 expression after intravenous, as well as intratumoral, injection. Moreover, the siRNA complex neither triggered the immune response nor changed blood-chemistry data, indicating its safety. These results suggested the potential of α-CDE (G3, DS2) as a novel siRNA-carrier candidate for both *in vivo* and *in vitro* applications.

#### **2.3. α-CDE (G3) as shRNA carriers**

of clathrin-dependent endocytosis inhibitors (i.e., chlorpromazine and sucrose) and raft-dependent endocytosis inhibitors (i.e., nystatin and filipin), but not by amiloride, the macropinocytosis inhibitor. These results suggested that the main mechanism of α-CDE (G3, DS2) uptake involved clathrin- and raft-dependent endocytosis. To confirm the release of the complex from endosomes, we observed the intracellular distribution of the α-CDE (G3, DS2)/FITC-pDNA complex by CLSM, finding that the green fluorescence originating from FITC-pDNA in the case of the α-CDE (G3, DS2) complex was predominantly localized in the cytoplasm to a much greater degree than that of the dendrimer system, confirming the improved capability for endosomal disruption conferred by the synergy between α-CDE and the proton-sponge effect of the dendrimer. Moreover, *in vivo* studies of α-CDE (G3, DS2), as well as dendrimer complexes with pDNA, were evaluated after intravenous administration of 50 μg pDNA/mouse at a charge ratio of 10. After 12 h, organs were collected, and pDNA levels in various organs were determined. The results showed that α-CDE (G3, DS2) delivered pDNA more efficiently in the liver and kidney; however, the highest gene-expression levels were observed in the spleen. Bloodchemistry data related to α-CDE administration, including aspartate aminotransferase (AST), lactate dehydrogenase (LDH), blood urea nitrogen (BUN), alanine aminotransferase (ALT), and creatinine (CRE) concentrations, showed minor changes as compared with those associated with the dendrimer [38]. These results suggested potential use of α-CDE (G3, DS2) as a safe and promising non-viral pDNA vector, although additional modifications of the α-CDE (G3, DS2) chemical structure were required to improve its nuclear translocation enable

Assessment of the siRNA-carrier ability of α-CDE (G3, DS2) was performed by Tsutsumi et al. using a luciferase-reporter system [40, 41]. Their results showed significant reductions in the luciferase activity of the α-CDE (G3, DS2) system as compared with that observed in the control and accompanied by negligible cytotoxicity after transfection with the ternary complex pDNA/siRNA/α-CDE (G3, DS2). In this study, α-CDE (G3, DS2) showed superior transfection efficiency relative to that of the dendrimer and other commercially available transfection reagents. Additionally, they observed that the complex localized exclusively to the cytoplasm, where RNAi-related activity occurs, and as a result of the lack of a nuclear-translocation moiety in α-CDE (G3, DS2). Tsutsumi et al. also reported efficient knockdown of the firefly luciferase gene using a α-CDE (G3, DS2)/siRNA binary system accompanied by negligible cytotoxicity as compared with the use of siRNA complexes with commercially available transfection reagent (Lipofectamine 2000 and RNAiFect). Furthermore, the physicochemical properties, local irritation, cytotoxicity, interferon response, cellular uptake, and intracellular distribution of the siRNA complexes, as well as the RNAi activity associated with the α-CDE (G3, DS2)/siRNA complex, were evaluated on endogenous gene-expression in Colon-26-luc and NIH3T3-luc cells stably expressing the pGL3 luciferase gene. The results revealed potent RNAi activity against Lamin A/C and Fas expression along with minor cytotoxicity as compared with commercial transfection agents [40]. Additionally, siRNA complexed with α-CDE (G3, DS2) was protected from degradation by serum nucleases. Intriguingly and somewhat

improved gene-expression results [39].

**2.2. α-CDE (G3) as siRNA carriers**

244 Cyclodextrin - A Versatile Ingredient

To improve upon and prolong the duration of RNAi-mediated gene knockdown, vectorbased shRNA-expression systems were developed [42]. Upon shRNA transfection into mammalian cells, the insert containing the vector is transferred to the nucleus, integrated into the host genome, expressed, and quickly processed by Dicer-dependent cleavage and loaded into the RNA-induced silencing complex, which is then directed to the target mRNA, resulting in its degradation. A previous study demonstrated the potential of α-CDE (G3, DS2) as a novel carrier of pDNA expressing shRNA [43]. In this study, the authors used pDNA expressing shRNA against the pGL3 firefly luciferase gene (shGL3). Even at a low charge ratio, α-CDE (G3, DS2) capably formed a stable condensed complex with shGL3 and induced the conformational transition of shRNA in solution from B-form to the more compact C-form DNA. Furthermore, α-CDE (G3, DS2) markedly inhibited shGL3 degradation by DNase I, and the α-CDE (G3, DS2)/shGL3 complex showed the most potent RNAi activity at a charge ratio of 20 along with negligible cytotoxicity and without off-target effects in A549 cells, a human alveolar adenocarcinoma cell line, while also transiently expressing the luciferase gene in NIH3T3-luc cells. Moreover, the addition of sufficient amounts of siGL3 along with α-CDE (G3, DS2)/shGL3 dramatically enhanced the RNAi activity, which was ascribed to the stabilizing effect of α-CDE (G3, DS2) against DNase I degradation of the shRNA accompanied by negligible cytotoxicity. These results suggested that α-CDE (G3, DS2) possessed the potential to be a novel shRNA carrier.

#### **2.4. Functionalized α-CDEs as cell-specific DNA and siRNA carriers**

In the early 1900s, the German Noble laureate Paul Ehrlich first introduced the concept of targeted drug delivery [44, 45]. At that time, he called it the "magic bullet", as it was able to deliver the drug specifically to microbes (such as bacteria) without harming the body. His continuous research eventually led to the development of the first effective drug against syphilis (Salvarsan). The primary aim of targeted drug-delivery systems is to increase the concentration of the medication in specific areas of the body relative to others, thereby improving its therapeutic index and reducing cytotoxicity. Various approaches have been adopted to target medications to the disease site [46, 47] with many compounds internalized inside of cells via receptor-mediated endocytosis. Receptor-mediated techniques use ligands attached to polyplexes to transfect cells with selected genes. Endocytosis is then mediated by various receptors, such as asialoglycoprotein receptor (ASGPR), mannose receptor (ManR), fucose receptor (FucR), and folate receptor (FR). However, despite the advantages offered by such systems, some drawbacks exist, including immune reactions against the carriers and rapid disposition of the carriers, as well as redistribution of the drugs after their release from the carriers [48]. Therefore, Arima et al. focused on how to improve CDEs for efficient delivery of gene and nucleic acid drugs to various organs through the attachment of various ligands to existing CDEs to aid the process of receptor-mediated endocytosis.

as pDNA vectors was decreased by the addition of FBS. Additionally, after co-incubation of Gal-α-CDE (G2, DSG4), dendrimer (G2), or Gal-α-CDE (G2, DS2) pDNA complex with asialofetuin and galactose, only a slight decrease in gene-transfer activity was observed in HepG2 cells, with no competitive effects. Consequently, these latter results confirmed that the mechanism associated with the enhanced gene-transfer activity of Gal-α-CDE (G2, DSG4) was not ASGPR-specific, but rather possibly due to other factors, such as increased stability of the pDNA complex or changes in intracellular trafficking. Collectively, these results suggested that Gal-α-CDE (G2, DSG4) exhibited enhanced pDNA-transport activity along with low cytotoxicity and considerable resistance to serum-associated degradation and could,

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As previously mentioned, Gal-α-CDE (G2, DSG4) did not exhibit hepatocyte-specific genedelivery activity. Therefore, Arima et al. prepared lactose-appended α-CDEs (Lac-α-CDEs) containing a glucose moiety as a spacer between the dendrimer and the lactose moiety [62, 63]. Of the various Lac-α-CDEs (G2) harboring different degrees of substitution of lactose (DSLs; 1.2, 2.6, 4.6, 6.2, and 10.2), Lac-α-CDE (G2, DSL2.6) exhibited the highest gene-transfer activity in HepG2 cells along with negligible cytotoxicity. To verify whether Lac-α-CDE (G2, DSL 2.6) could bind to galactose-binding lectins, the association constant of Lac-α-CDE (G2, DSL2.6) with peanut lectin was determined and compared with that of α-CDE (G2, DS2) using surface plasmon resonance. The results showed that the association constant of Lac-α-CDE (G2, DSL2.6) was 100-fold greater than that of α-CDE (G2, DS2). It was previously reported that the dissociation constant of asialofetuin for ASGPR located on HepG2 cells is ~3.61 × 10−9 M [64]. These results indicated that the specific galactose-binding ability of Lac-α-CDE (G2, DSL2.6) was maintained, although the magnitude was not as strong as that of asialofetuin. To confirm the ASGPR-mediated gene-transfer activity of Lac-α-CDE (G2, DSL2.6), the effects of asialofetuin, as a competitor on this activity, was examined in HepG2 cells. The results revealed that the gene-transfer activity of Lac-α-CDE (G2, DSL2.6) was significantly suppressed by asialofetuin, and that cellular association of the Lac-α-CDE (G2, DSL2.6)/Alexa-pDNA complex was markedly reduced in the presence of asialofetuin. However, no inhibitory effect of asialofetuin was observed on the activity of α-CDE (G2, DS2)/Alexa-pDNA in HepG2 cells. These results indicated that the gene-transfer activity of Lac-α-CDE (G2, DSL2.6) was mediated by ASGPR endocytosis. Arima et al. then evaluated the ability of Lac-α-CDE (G2, DSL2.6) to deliver pDNA *in vivo* using the pGL3 luciferase system in mice. The complexes were intravenously injected into mice, and after 12 h, luciferase activity was determined. They observed that luciferase activity in the Lac-α-CDE (G2, DSL2.6) system was significantly higher than that observed in the α-CDE (G2) system in the liver. Furthermore, to estimate the safety profile of Lac-α-CDE (G2, DSL2.6), the effect of its pDNA complex on blood-chemistry data, such as CRE, BUN, AST, ALT, and LDH concentrations, was analyzed following intravenous administration to mice. The ALT concentration in the Lac-α-CDE (G2, DSL2.6) system, as well as in the jetPEI-hepatocyte system, was slightly elevated (no significant difference) as compared with that observed in the control. By contrast, all other parameters in the Lac-α-CDE (G2, DSL2.6) system were almost equivalent to those of controls. These results strongly suggested that Lac-α-CDE (G2, DSL2.6) exhibited hepatocyte-specific gene-transfer activity along with a good safety profile *in vivo*.

therefore, represent an excellent non-viral gene-delivery carrier.

*2.4.2. Lactosylated α-CDEs as hepatocyte-selective pDNA and siRNA carriers*

#### *2.4.1. Galactosylated α-CDE as a hepatocyte-selective pDNA carrier*

The liver consists mainly of hepatocytes (nearly 70%) and parenchymal cells. Gene- or drugtargeting systems designed to target the liver are usually directed to hepatocytes, which overexpress ASGPRs on their cell surface. These ASGPRs mediate the removal of potentially hazardous glycoconjugates from the blood; therefore, ASGPRs are usually targeted using galactose residues coupled with a core molecule to enhance binding [49, 50]. Many approaches, including but not limited to (1) intravenous injection of pDNA within liposomes [51, 52] or via ASGPR targeting [53] and (2) intra-portal injection of recombinant adenovirus [54] and retroviral vectors [55] have been adopted to deliver foreign genes *in vivo* to hepatocytes. Moreover, an *in vitro* system that takes advantage of ASGPR-mediated endocytosis to transfect hepatocytes with an exogenous DNA using a soluble DNA carrier was developed [56]. Various ASGPR-mediated gene-delivery systems using different polymers have also been described, including galactose-polyethylene glycol (PEG)-poly(L-lysine) [57], galactosylated PEG-graft-polyethylenimine (PEI) [58, 59], and galactosylated chitosan-grafted-PEI [60]. In order to attain hepatocyte-specificity and/or improve the efficacy of α-CDE as a genedelivery carrier, Arima et al. attached a galactose residue to form Gal-α-CDE (G2) as a novel non-viral carrier [61]. The galactose moieties were attached to the primary amino groups of α-CDE (G2) through a spacer consisting of α-D-galctopyranosylphenyl isothiocyanate, achieving various degrees of substitutions of galactose (DSGs; 1, 4, 5, 8, and 15). Evaluation of the complexation ability of the pDNA complexes by electrophoresis showed that the Galα-CDEs (G2) could form complexes with pDNA; however, complexation ability decreased along with increasing DSG values, possibly due to decreases in free positive-charged primary amino groups. Moreover, the ability of these carriers to protect the pDNA from degradation by serum nucleases also decreased along with increasing DSGs, likely due to the attenuated interactions and loss of pDNA-condensation ability in the presence of high DSGs in the conjugates. Additionally, they observed that Gal-α-CDEs (G2, DSG4) elicited the most prominent gene-transfer activity relative to that of the dendrimer (G2), and α-CDE (G2, DS2) in HepG2 cells, a human hepatoma cell line, NIH3T3 cells, and A549 cells showed no cytotoxicity up to a charge ratio of 200. Surprisingly, this was independent of ASGPR expression, possibly due to the inability of the spacer to properly present the galactose residue for receptor recognition. Therefore, these results suggested the potential of Gal-α-CDE (G2, DSG4) as a novel non-viral vector independent of cell-surface ASGPR expression. It is worth mentioning that in this study, the authors used a cancer-cell line (HepG2) and not normal hepatocytes, given that they both express ASGPR at similar levels, and HepG2 cells are widely used by scientists engaged in targeting studies for genes using non-viral carriers. Surprisingly, the addition of 10% fetal bovine serum (FBS) did not alter the gene transfer activity of Gal-α-CDE (G2, DSG4); however, this activity on the part of the dendrimer (G2), as well as that of α-CDE (G2, DSG2), as pDNA vectors was decreased by the addition of FBS. Additionally, after co-incubation of Gal-α-CDE (G2, DSG4), dendrimer (G2), or Gal-α-CDE (G2, DS2) pDNA complex with asialofetuin and galactose, only a slight decrease in gene-transfer activity was observed in HepG2 cells, with no competitive effects. Consequently, these latter results confirmed that the mechanism associated with the enhanced gene-transfer activity of Gal-α-CDE (G2, DSG4) was not ASGPR-specific, but rather possibly due to other factors, such as increased stability of the pDNA complex or changes in intracellular trafficking. Collectively, these results suggested that Gal-α-CDE (G2, DSG4) exhibited enhanced pDNA-transport activity along with low cytotoxicity and considerable resistance to serum-associated degradation and could, therefore, represent an excellent non-viral gene-delivery carrier.

#### *2.4.2. Lactosylated α-CDEs as hepatocyte-selective pDNA and siRNA carriers*

receptor (FucR), and folate receptor (FR). However, despite the advantages offered by such systems, some drawbacks exist, including immune reactions against the carriers and rapid disposition of the carriers, as well as redistribution of the drugs after their release from the carriers [48]. Therefore, Arima et al. focused on how to improve CDEs for efficient delivery of gene and nucleic acid drugs to various organs through the attachment of various ligands to

The liver consists mainly of hepatocytes (nearly 70%) and parenchymal cells. Gene- or drugtargeting systems designed to target the liver are usually directed to hepatocytes, which overexpress ASGPRs on their cell surface. These ASGPRs mediate the removal of potentially hazardous glycoconjugates from the blood; therefore, ASGPRs are usually targeted using galactose residues coupled with a core molecule to enhance binding [49, 50]. Many approaches, including but not limited to (1) intravenous injection of pDNA within liposomes [51, 52] or via ASGPR targeting [53] and (2) intra-portal injection of recombinant adenovirus [54] and retroviral vectors [55] have been adopted to deliver foreign genes *in vivo* to hepatocytes. Moreover, an *in vitro* system that takes advantage of ASGPR-mediated endocytosis to transfect hepatocytes with an exogenous DNA using a soluble DNA carrier was developed [56]. Various ASGPR-mediated gene-delivery systems using different polymers have also been described, including galactose-polyethylene glycol (PEG)-poly(L-lysine) [57], galactosylated PEG-graft-polyethylenimine (PEI) [58, 59], and galactosylated chitosan-grafted-PEI [60]. In order to attain hepatocyte-specificity and/or improve the efficacy of α-CDE as a genedelivery carrier, Arima et al. attached a galactose residue to form Gal-α-CDE (G2) as a novel non-viral carrier [61]. The galactose moieties were attached to the primary amino groups of α-CDE (G2) through a spacer consisting of α-D-galctopyranosylphenyl isothiocyanate, achieving various degrees of substitutions of galactose (DSGs; 1, 4, 5, 8, and 15). Evaluation of the complexation ability of the pDNA complexes by electrophoresis showed that the Galα-CDEs (G2) could form complexes with pDNA; however, complexation ability decreased along with increasing DSG values, possibly due to decreases in free positive-charged primary amino groups. Moreover, the ability of these carriers to protect the pDNA from degradation by serum nucleases also decreased along with increasing DSGs, likely due to the attenuated interactions and loss of pDNA-condensation ability in the presence of high DSGs in the conjugates. Additionally, they observed that Gal-α-CDEs (G2, DSG4) elicited the most prominent gene-transfer activity relative to that of the dendrimer (G2), and α-CDE (G2, DS2) in HepG2 cells, a human hepatoma cell line, NIH3T3 cells, and A549 cells showed no cytotoxicity up to a charge ratio of 200. Surprisingly, this was independent of ASGPR expression, possibly due to the inability of the spacer to properly present the galactose residue for receptor recognition. Therefore, these results suggested the potential of Gal-α-CDE (G2, DSG4) as a novel non-viral vector independent of cell-surface ASGPR expression. It is worth mentioning that in this study, the authors used a cancer-cell line (HepG2) and not normal hepatocytes, given that they both express ASGPR at similar levels, and HepG2 cells are widely used by scientists engaged in targeting studies for genes using non-viral carriers. Surprisingly, the addition of 10% fetal bovine serum (FBS) did not alter the gene transfer activity of Gal-α-CDE (G2, DSG4); however, this activity on the part of the dendrimer (G2), as well as that of α-CDE (G2, DSG2),

existing CDEs to aid the process of receptor-mediated endocytosis.

*2.4.1. Galactosylated α-CDE as a hepatocyte-selective pDNA carrier*

246 Cyclodextrin - A Versatile Ingredient

As previously mentioned, Gal-α-CDE (G2, DSG4) did not exhibit hepatocyte-specific genedelivery activity. Therefore, Arima et al. prepared lactose-appended α-CDEs (Lac-α-CDEs) containing a glucose moiety as a spacer between the dendrimer and the lactose moiety [62, 63]. Of the various Lac-α-CDEs (G2) harboring different degrees of substitution of lactose (DSLs; 1.2, 2.6, 4.6, 6.2, and 10.2), Lac-α-CDE (G2, DSL2.6) exhibited the highest gene-transfer activity in HepG2 cells along with negligible cytotoxicity. To verify whether Lac-α-CDE (G2, DSL 2.6) could bind to galactose-binding lectins, the association constant of Lac-α-CDE (G2, DSL2.6) with peanut lectin was determined and compared with that of α-CDE (G2, DS2) using surface plasmon resonance. The results showed that the association constant of Lac-α-CDE (G2, DSL2.6) was 100-fold greater than that of α-CDE (G2, DS2). It was previously reported that the dissociation constant of asialofetuin for ASGPR located on HepG2 cells is ~3.61 × 10−9 M [64]. These results indicated that the specific galactose-binding ability of Lac-α-CDE (G2, DSL2.6) was maintained, although the magnitude was not as strong as that of asialofetuin. To confirm the ASGPR-mediated gene-transfer activity of Lac-α-CDE (G2, DSL2.6), the effects of asialofetuin, as a competitor on this activity, was examined in HepG2 cells. The results revealed that the gene-transfer activity of Lac-α-CDE (G2, DSL2.6) was significantly suppressed by asialofetuin, and that cellular association of the Lac-α-CDE (G2, DSL2.6)/Alexa-pDNA complex was markedly reduced in the presence of asialofetuin. However, no inhibitory effect of asialofetuin was observed on the activity of α-CDE (G2, DS2)/Alexa-pDNA in HepG2 cells. These results indicated that the gene-transfer activity of Lac-α-CDE (G2, DSL2.6) was mediated by ASGPR endocytosis. Arima et al. then evaluated the ability of Lac-α-CDE (G2, DSL2.6) to deliver pDNA *in vivo* using the pGL3 luciferase system in mice. The complexes were intravenously injected into mice, and after 12 h, luciferase activity was determined. They observed that luciferase activity in the Lac-α-CDE (G2, DSL2.6) system was significantly higher than that observed in the α-CDE (G2) system in the liver. Furthermore, to estimate the safety profile of Lac-α-CDE (G2, DSL2.6), the effect of its pDNA complex on blood-chemistry data, such as CRE, BUN, AST, ALT, and LDH concentrations, was analyzed following intravenous administration to mice. The ALT concentration in the Lac-α-CDE (G2, DSL2.6) system, as well as in the jetPEI-hepatocyte system, was slightly elevated (no significant difference) as compared with that observed in the control. By contrast, all other parameters in the Lac-α-CDE (G2, DSL2.6) system were almost equivalent to those of controls. These results strongly suggested that Lac-α-CDE (G2, DSL2.6) exhibited hepatocyte-specific gene-transfer activity along with a good safety profile *in vivo*.

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

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

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

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

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

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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

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

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

*carriers*

for nucleic acid delivery [68].

KC-selective siRNA carrier.

for ManR, possibly due to the rigidity of the spacer.

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

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