In Vitro Gene Delivery Mediated by Asialofetuin-Appended Cationic Liposomes Associated with γ-Cyclodextrin into Hepatocytes

The purpose of this study is to evaluate in vitro gene delivery mediated by asialofetuin-appended cationic liposomes (AF-liposomes) associating cyclodextrins (CyD/AF-liposomes) as a hepatocyte-selective nonviral vector. Of various CyDs, AF-liposomes associated with plasmid DNA (pDNA) and γ-cyclodextrin (γ-CyD) (pDNA/γ-CyD/AF-liposomes) showed the highest gene transfer activity in HepG2 cells without any significant cytotoxicity. In addition, γ-CyD enhanced the encapsulation ratio of pDNA with AF-liposomes, and also increased gene transfer activity as the entrapment ratio of pDNA into AF-liposomes was increased. γ-CyD stabilized the liposomal membrane of AF-liposomes and inhibited the release of calcein from AF-liposomes. The stabilizing effect of γ-CyD may be, at least in part, involved in the enhancing gene transfer activity of pDNA/γ-CyD/AF-liposomes. Therefore, these results suggest the potential use of γ-CyD for an enhancer of transfection efficiency of AF-liposomes

Potential Use of Folate-polyethylene glycol (PEG)-Appended Dendrimer (G3) Conjugate with alpha-Cyclodextrin as DNA Carriers to Tumor Cells

We previously reported that polyamidoamine STARBURST dendrimer (generation 3, G3) (dendrimer) conjugate with alpha-cyclodextrin (alpha-CyD) having an average degree of substitution of 2.4 of alpha-CyD (alpha-CDE) provided remarkable aspects as novel carriers for DNA and siRNA. To develop novel alpha-CDE derivatives with tumor cell specificity, we prepared folate-appended alpha-CDEs (Fol-alpha-CDEs) and folate-polyethylene glycol (PEG)-appended alpha-CDEs (Fol-PalphaCs) with the various degrees of substitution of folate (DSF), and evaluated in vitro and in vivo gene transfer activity, cytotoxicity, cellular association and physicochemical properties. In vitro gene transfer activity of Fol-alpha-CDEs (G3, DSF 2, 5 or 7) was lower than that of α-CDE (G3) in KB cells, folate receptor (FR)-overexpressing cancer cells. Of the three Fol-PalphaCs (G3, DSF 2, 5 or 7), Fol-PalphaC (G3, DSF 5) had the highest gene transfer activity in KB cells. The activity of Fol-PalphaC (G3, DSF 5) was significantly higher than that of alpha-CDE (G3) in KB cells, but not in A549 cells, FR-negative cells. Negligible cytotoxicity of the pDNA complex with Fol-PalphaC (G3, DSF 5) was observed in KB cells or A549 cells up to a charge ratio of 100/1 (carrier/pDNA). The cellular association of the pDNA complex with Fol-PalphaC (G3, DSF 5) could be mediated by FR on KB cells, resulting in its efficient cellular uptake. Fol-PalphaC (G3, DSF 5) had higher binding affinity with folate binding protein (FBP) than alpha-CDE (G3), although the physicochemical properties of pDNA complex with Fol-PalphaC (G3, DSF 5) were almost comparable to that with alpha-CDE (G3), although the onset charge ratio and the compaction ability of Fol-PalphaC (G3, DSF 5) were slightly different. Fol-PalphaC (G3, DSF 5) tended to show higher gene transfer activity than alpha-CDE (G3) 12 h after intratumoral administration in mice. These results suggest that Fol-PalphaC (G3, DSF 5), not Fol-alpha-CDEs, could be potentially used as a FR-overexpressing cancer cell-selective DNA carrier

Potential Use of C

The photosensitizing ability of C60/2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) nanoparticles under visible light irradiation was studied by electron spin resonance (ESR) and phototoxicity on cancer cells. In addition, the photoinduced antitumor effect to the tumor-bearing mice was evaluated. C60 nanoparticles were prepared by grinding a mixture of HP-β-CyD. The resulting C60/HP-β-CyD nanoparticles were highly-sensitive to visible light and generated higher levels of 1O2 than protoporphyrin IX (PpIX). C60/HP-β-CyD reduced the viability of cancer cells (HeLa cells and A549 cells) in response to irradiation by visible light in a dose-dependent manner. The IC50 values of the C60/HP-β-CyD nanoparticles was 10 μM for HeLa cells and 60 μM for A549 cells at an irradiation level of 35 mW/cm2. The photodynamic effect of C60/HP-β-CyD nanoparticles on the in vivo growth of mouse sarcoma S-180 cells was evaluated after intratumor injection. The outcome of PDT by C60/HP-β-CyD was directly dependent on the dose of irradiated light. Treatment with C60/HP-β-CyD nanoparticles at a C60 dose of 2.0 mg/kg under visible light irradiation at 350 mW/cm2 (63 J/cm2) markedly suppressed tumor growth, whereas that at 30 J/cm2 was less effective. These findings suggest that C60/HP-β-CyD nanoparticles represent a promising candidate for use in cancer treatment by PDT

Evaluation of polyamidoamine dendrimer/α-cyclodextrin conjugate (generation 3, G3) as a novel carrier for small interfering RNA (siRNA)

As the first step toward an evaluation of the potential use of the PAMAM dendrimer (G3) conjugate with α-cyclodextrin (α-CDE) for a small interfering RNA (siRNA) carrier, the ternary complexes of α-CDE or the transfection reagents such as LipofactamineTM2000 (L2), TransFastTM (TF) and LipofectinTM (LF) with plasmid DNA (pDNA) and siRNA were prepared, and their RNAi effects, cytotoxicity, physicochemical properties and intracellular distribution were compared. Here the pGL2- control vector (pGL2) and pGL3-control vector (pGL3) encoding the firefly luciferase gene and the two corresponding siRNAs (siGL2 and siGL3) were used. The ternary complexes of pGL3/siGL3/α-CDE showed the potent RNAi effects with negligible cytotoxicity compared to those of the transfection reagents in various cells. α-CDE strongly interacted with both pDNA and siRNA, and suppressed siRNA degradation by serum, compared to those of the transfection reagents. α-CDE allowed fluorescent labeled siRNA to distribute in cytoplasm, whereas the transfection reagents resided in both nucleus and cytoplasm in NIH3T3 cells. Furthermore, the binary complex of siRNA/α-CDE provided the significant RNAi effect in NIH3T3 cells transiently and stably expressing luciferase gene. These results suggest that α-CDE may be utilized as a novel carrier for siRNA

Antioxidant and renoprotective activity of chitosan in nephrectomized rats.

The effect of chitosan on oxidative stress and chronic renal failure was investigated using 5/6 nephrectomized rats. The ingestion of chitosan over a 4-week period resulted in a significant decrease in total body weight, glucose, serum creatinine and indoxyl sulfate levels (P=0.0011, P=0.0006, P=0.0012, and P=0.0005, respectively), compared with the non-treated nephrectomized group. The ingestion of chitosan also resulted in a lowered ratio of oxidized to reduced albumin (P=0.003) and an increase in biological antioxidant potential (P=0.023). Interestingly, the oxidized albumin ratio was correlated with serum indoxyl sulfate levels in vivo. These results suggest that the ingestion of chitosan results in a significant reduction in the levels of pro-oxidants, such as uremic toxins, in the gastrointestinal tract, thereby inhibiting the subsequent development of oxidative stress in the systemic circulation.The effect of chitosan on oxidative stress and chronic renal failure was investigated using 5/6 nephrectomized rats. The ingestion of chitosan over a 4-week period resulted in a significant decrease in total body weight, glucose, serum creatinine and indoxyl sulfate levels (P=0.0011, P=0.0006, P=0.0012, and P=0.0005, respectively), compared with the non-treated nephrectomized group. The ingestion of chitosan also resulted in a lowered ratio of oxidized to reduced albumin (P=0.003) and an increase in biological antioxidant potential (P=0.023). Interestingly, the oxidized albumin ratio was correlated with serum indoxyl sulfate levels in vivo. These results suggest that the ingestion of chitosan results in a significant reduction in the levels of pro-oxidants, such as uremic toxins, in the gastrointestinal tract, thereby inhibiting the subsequent development of oxidative stress in the systemic circulation

Uekama K. Contribution of P-glycoprotein to the enhancing effects of dimethyl-beta-cyclodextrin on oral bioavailability of tacrolimus

ABSTRACT We recently reported that of all hydrophilic cyclodextrin (CyD) derivatives examined, 2,6-di-O-methyl-␤-cyclodextrin (DM-␤-CyD) most significantly increased the aqueous solubility and the dissolution rate, resulting in the improvement of oral bioavailability of the immunosuppressive drug tacrolimus in rats. In the present study, we showed that DM-␤-CyD increased the dissolution rate and oral bioavailability of tacrolimus in rats with increases in the molar ratio of the complexes (DM-␤-CyD: tacrolimus). However, nonlinear pharmacokinetic behavior of tacrolimus after oral administration in rats was observed. Thus, an additional mechanism of the solubilizing effect of DM-␤-CyD on oral bioavailability of tacrolimus was postulated. To gain insight into this additional mechanism of action of DM-␤-CyD, its effects on the efflux of tacrolimus and rhodamine 123, a P-glycoprotein (P-gp) substrate, were examined using both Caco-2 and vinblastine-resistant Caco-2 (Caco-2R) cell monolayers. Pretreatment of the apical membranes of the monolayers with DM-␤-CyD decreased the efflux of tacrolimus and rhodamine 123 without an associated cytotoxicity. DM-␤-CyD decreased the P-gp level in the apical membranes of both Caco-2 and Caco-2R cell monolayers, probably by allowing release of P-gp from the apical membrane into the transport buffer. DM-␤-CyD, however, did not decrease the MDR1 gene expression in Caco-2 or Caco-2R cells. These results suggested that the enhancing effect of DM-␤-CyD on the oral bioavailability of tacrolimus is due not only to its solubilizing effect but also, at least in part, to its inhibitory effect on the P-gp-mediated efflux of tacrolimus from intestinal epithelial cells

Surface-Deacetylated Chitin Nano-Fiber/Hyaluronic Acid Composites as Potential Antioxidative Compounds for Use in Extended-Release Matrix Tablets

In this study, we examined a possible use of a surface-deacetylated chitin nano-fiber (SDCH-NF) and hyaluronic acid (HA) interpolymer complex (IPC) tablet as a potential antioxidative compound in extended-release matrix tablets. The antioxidant properties of untreated chitin (UCH), SDCH-NF, and HA were examined using N-centered radicals derived from 1,1′-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). SDCH-NF and HA had acceptable scavenging abilities and were relatively efficient radical scavengers, but UCH was much less effective. The results suggest that SDCH-NF and HA could serve as scavengers of compounds related to the development of oxidative stress. An SDCH-NF/HA IPC tablet was prepared and evaluated as an extended-release tablet matrix using famotidine (FMT) as a model drug. The release of FMT from the IPC tablet (DCF-NF:HA = 1:1) was slower than that from a SDCH-NF only tablet. Turbidity measurements and X-ray diffraction (XRD) data also indicated that the optimum complexation ratio for IPC between SDCH-NF/HA is 1/1, resulting in a good relationship between turbidity or XRD of the complex and the release ratio of FMT. These results suggest that an SDCH-NF/HA tablet has the potential for use in an extended-release IPC tablet with a high antioxidant activity