In Vitro and In Vivo Co-delivery of siRNA and Doxorubicin by Folate-PEG-Appended Dendrimer/Glucuronylglucosyl-ß-Cyclodextrin Conjugate.

We have previously reported the utility of folate-polyethylene glycol-appended dendrimer conjugate with glucuronylglucosyl-ß-cyclodextrin (Fol-PEG-GUG-ß-CDE) (generation 3) as a tumor-selective carrier for siRNA against polo-like kinase 1 (siPLK1) in vitro. In the present study, we evaluated the potential of Fol-PEG-GUG-ß-CDE as a carrier for the low-molecular antitumor drug doxorubicin (DOX). Further, to fabricate advanced antitumor agents, we have prepared a ternary complex of Fol-PEG-GUG-ß-CDE/DOX/siPLK1 and evaluated its antitumor activity both in vitro and in vivo. Fol-PEG-GUG-ß-CDE released DOX in an acidic pH and enhanced the cellular accumulation and cytotoxic activity of DOX in folate receptor-α (FR-α)-overexpressing KB cells. Importantly, the Fol-PEG-GUG-ß-CDE/DOX/siPLK1 ternary complex exhibited higher cytotoxic activity than a binary complex of Fol-PEG-GUG-ß-CDE with DOX or siPLK1 in KB cells. In addition, the cytotoxic activity of the ternary complex was reduced by the addition of folic acid, a competitor against FR-α. Furthermore, the ternary complex showed a significant antitumor activity after intravenous administration to the tumor-bearing mice. These results suggest that Fol-PEG-GUG-ß-CDE has the potential of a tumor-selective co-delivery carrier for DOX and siPLK1.

Ternary complexes of folate-PEG-appended dendrimer (G4)/α-cyclodextrin conjugate, siRNA and low-molecular-weight polysaccharide sacran as a novel tumor-selective siRNA delivery system.

We previously developed a tumor-selective siRNA carrier by preparing polyamidoamine dendrimer (generation 4, G4) conjugates with α-cyclodextrin and folate-polyethylene glycol (Fol-PαC (G4)). In the present study, we developed ternary complexes of Fol-PαC (G4)/siRNA with low-molecular-weight-sacrans to achieve more effective siRNA transfer activity. Among the different molecular-weight sacrans, i.e. sacran 100, 1000 and 10,000 (MW 44,889Da, 943,692Da and 1,488,281Da, respectively), sacran 100 significantly increased the cellular uptake and the RNAi effects of Fol-PαC (G4)/siRNA binary complex with negligible cytotoxicity in KB cells (folate receptor-α positive cells). In addition, the ζ-potential and particle size of Fol-PαC (G4)/siRNA complex were decreased by the ternary complexation with sacran 100. Importantly, the in vivo RNAi effect of the ternary complex after the intravenous administration to tumor-bearing BALB/c mice was significantly higher than that of the binary complex. In conclusion, Fol-PαC (G4)/siRNA/sacran 100 ternary complex has a potential as a novel tumor-selective siRNA delivery system.

In Vitro and In Vivo Tumor-Targeting siRNA Delivery Using Folate-PEG-appended Dendrimer (G4)/α-Cyclodextrin Conjugates.

We previously reported that folate-polyethylene glycol (PEG)-appended dendrimer (generation 3)/α-cyclodextrin conjugate (Fol-PαC (G3)) shows folate receptor-α (FR-α)-overexpressing tumor cell-selective in vitro siRNA transfer activity. However, Fol-PαC (G3)/siRNA complex did not induce a significant in vivo RNAi effect after intravenous administration to tumor-bearing mice, possibly resulting from immediate dissociation of the complex in blood. Herein, to develop the novel siRNA carrier having high blood circulating ability, high in vivo siRNA transfer activity, and high safety profile, we newly prepared Fol-PαCs with higher generation (G4) and evaluated their potential as tumor-targeting siRNA carriers in vitro and in vivo. Fol-PαC (G4, average degree of substitution of α-cyclodextrin (DSC) 2.9, average degree of substitution of folate-PEG (DSF) 2)/siRNA complex had the prominent RNAi effect through adequate physicochemical properties, FR-α-mediated endocytosis, efficient endosomal escape, and siRNA delivery to cytoplasm with negligible cytotoxicity. Importantly, Fol-PαC (G4, DSC2.9, DSF2) improved the serum stability, blood circulating ability, and in vivo RNAi effects of siRNA, compared to Fol-PαC (G3). Furthermore, Fol-PαC (G4, DSC2.9, DSF2) complex with siRNA against Polo-like kinase 1 (siPLK1) suppressed the tumor growth compared to control siRNA complex. These results suggest that Fol-PαC (G4, DSC2.9, DSF2) has the potential as a novel tumor-targeting siRNA carrier in vitro and in vivo.

Design and evaluation of thioalkylated mannose-modified dendrimer (G3)/α-cyclodextrin conjugates as antigen-presenting cell-selective siRNA carriers.

To design and evaluate the potential use of thioalkylated mannose-modified dendrimer (generation 3; G3) conjugates with α-cyclodextrin (Man-S-α-CDE (G3)) as novel antigen-presenting cell (APC)-selective siRNA carriers, we investigated the RNAi effects of siRNA complexes with Man-S-α-CDEs (G3). Man-S-α-CDE (G3, average degree of substitution of mannose (DSM) 4)/siRNA complex had the potent RNAi effects in both NR8383 cells, a rat alveolar macrophage cell line, and JAWSII cells, a mouse dendritic cell line, through adequate physicochemical properties, mannose receptor (MR)-mediated cellular uptake, and efficient phagosomal escape of the siRNA complex. In addition, cytotoxic activities of the siRNA complexes with α-CDE (G3, DS2) and Man-S-α-CDE (G3, DSM4) were almost negligible up to a charge ratio of 100 (carrier/siRNA). Taken together, these results suggest that Man-S-α-CDE (G3, DSM4) has the potential for a novel APC-selective siRNA carrier.

Preparation and evaluation of polyamidoamine dendrimer conjugate with glucuronylglucosyl-ß-cyclodextrin (G3) as a novel carrier for siRNA.

Abstract In this study, we newly synthesized the polyamidoamine STARBURST dendrimer (dendrimer, generation 3: G3) conjugates with 6-O-α-(4-O-α-D-glucuronyl)-D-glucosyl-ß-cyclodextrin [GUG-ß-CDE (G3)] having the various degrees of substitution (DS) of GUG-ß-cyclodextrin of 1.6, 3.0, 3.7, 5.0 and 8.6, and evaluated them as a siRNA transfer carrier. GUG-ß-CDEs (G3) formed the positively charged and nano-order complexes with siRNA. Of the siRNA complexes with five GUG-ß-CDEs (G3), the complex with GUG-ß-CDE (G3, DS 3.7) showed the highest RNAi effect and cellular uptake with negligible cytotoxicity in KB cells at a charge ratio of 20. In addition, the RNAi effect and cellular uptake of the complex with GUG-ß-CDE (G3, DS 3.7) were higher than those of α-CDE (G3, DS 2.4) and comparable to those of Lipofectamine™ 2000. Furthermore, the complex with GUG-ß-CDE (G3, DS 3.7) possessed the endosomal escaping ability, the releasing property of siRNA in the cytoplasm and serum resistance. These results suggest that GUG-ß-CDE (G3, DS 3.7) has the potential as a novel siRNA carrier.

Potential use of fucose-appended dendrimer/α-cyclodextrin conjugates as NF-κB decoy carriers for the treatment of lipopolysaccharide-induced fulminant hepatitis in mice.

The purpose of the present study is to treat lipopolysaccharide (LPS)-induced fulminant hepatitis by NF-κB decoy complex with fucose-appended dendrimer (generation 2; G2) conjugate with α-cyclodextrin (Fuc-S-α-CDE (G2)). Fuc-S-α-CDE (G2, average degree of substitution of fucose (DSF2))/NF-κB decoy complex significantly suppressed nitric oxide and tumor necrosis factor-α (TNF-α) production from LPS-stimulated NR8383 cells, a rat alveolar macrophage cell line, by adequate physicochemical properties and fucose receptor-mediated cellular uptake. Intravenous injection of Fuc-S-α-CDE (G2, DSF2)/NF-κB decoy complex extended the survival of LPS-induced fulminant hepatitis model mice. In addition, Fuc-S-α-CDE (G2, DSF2)/NF-κB decoy complex administered intravenously highly accumulated in the liver, compared to naked NF-κB decoy alone. Furthermore, the liver accumulation of Fuc-S-α-CDE (G2, DSF2)/NF-κB decoy complex was inhibited by the pretreatment with GdCl3, a specific inhibitor of Kupffer cell uptake. Also, the serum aspartate aminotransferase, alanine aminotransferase and TNF-α levels in LPS-induced fulminant hepatitis model mice were significantly attenuated by the treatment with Fuc-S-α-CDE (G2, DSF2)/NF-κB decoy complex, compared with naked NF-κB decoy alone. Taken together, these results suggest that Fuc-S-α-CDE (G2, DSF2) has the potential for a novel Kupffer cell-selective NF-κB decoy carrier for the treatment of LPS-induced fulminant hepatitis in mice.

Involvement of autophagy in antitumor activity of folate-appended methyl-ß-cyclodextrin.

Autophagy, the major lysosomal pathway for recycling intracellular components including organelles, is emerging as a key process regulating tumorigenesis and cancer therapy. Most recently, we newly synthesized folate-appended methyl-ß-cyclodextrin (FA-M-ß-CyD), and demonstrated the potential of FA-M-ß-CyD as a new antitumor drug. In this study, we investigated whether anticancer activity of FA-M-ß-CyD in folate receptor-α (FR-α)-positive tumor cells is involved in autophagy. In contrast to methyl-ß-cyclodextrin (M-ß-CyD), FA-M-ß-CyD entered KB cells (FR-α (+)) through CLIC/GEEC endocytosis. No significant depression in the DNA content was observed in KB cells after treatment with FA-M-ß-CyD. Additionally, the transmembrane potential of mitochondria after treatment with FA-M-ß-CyD was drastically elevated. Meanwhile, FA-M-ß-CyD induced the formation of autophagic vacuoles, which were partially colocalized with mitochondria, in KB cells. Taken together, these results suggest that FR-α-expressing cell-selective cytotoxic activity of FA-M-ß-CyD could be mediated by the regulation of autophagy, rather than the induction of apoptosis.

Folate-PEG-appended dendrimer conjugate with α-cyclodextrin as a novel cancer cell-selective siRNA delivery carrier.

We previously reported that of the various polyamidoamine (PAMAM) STARBURST dendrimer (generation 3, G3) (dendrimer) conjugates with cyclodextrins (CyDs), the dendrimer (G3) conjugate with α-CyD having an average degree of substitution of 2.4 (α-CDE (G3)) has the greatest potential for a novel carrier for siRNA in vitro and in vivo. To improve the siRNA transfer activity and the lack of target specificity of α-CDE (G3), we prepared folate-polyethylene glycol (PEG)-appended α-CDEs (G3) (Fol-PαCs) with various degrees of substitution of folate (DSF) and evaluated their siRNA transfer activity to folate receptor (FR)-overexpressing cancer cells in vitro and in vivo. Of the three Fol-PαCs (G3, DSF 2, 4 and 7), Fol-PαC (G3, DSF 4) had the highest siRNA transfer activity in KB cells (FR-positive). Fol-PαC (G3, DSF 4) was endocytosed into KB cells through FR. No cytotoxicity of the siRNA complex with Fol-PαC (G3, DSF 4) was observed in KB cells (FR-positive) or A549 cells (FR-negative) up to the charge ratio of 100/1 (carrier/siRNA). In addition, the siRNA complex with Fol-PαC (G3, DSF 4) showed neither interferon response nor inflammatory response. Importantly, the siRNA complex with Fol-PαC (G3, DSF 4) tended to show the in vivo RNAi effects after intratumoral injection and intravenous injection in tumor cells-bearing mice. The FITC-labeled siRNA and TRITC-labeled Fol-PαC (G3, DSF 4) were actually accumulated in tumor tissues after intravenous injection in the mice. In conclusion, the present results suggest that Fol-PαC (G3, DSF 4) could potentially be used as a FR-overexpressing cancer cell-selective siRNA delivery carrier in vitro and in vivo.