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[Display omitted] • Novel sustained release system for protein drugs was developed exploiting the "astringency" mechanisms. • This sustained release system is prepared by only mixing with tannic acid. • This sustained release system is available for various protein drugs. In recent years, protein drug development has gained momentum, and simple and facile controlled-release systems without loss of activity are required. Herein, we developed a sustained-release system for protein drugs by exploiting the "astringency" mechanism, namely insoluble precipitate formation by interacting with tannic acid. Tannic acid formed insoluble precipitates with various protein drugs, such as nisin, insulin, lysozyme, ovalbumin, hyaluronidase, and human immunoglobulin G, through hydrophobic interactions and hydrogen bonds. The lysozyme/tannic acid complex retained in vitro lytic activity. Precipitates of the insulin/tannic acid complex prolonged hypoglycemic effects without loss of activity after subcutaneous administration. The ovalbumin/tannic acid complex enhanced anti-ovalbumin antibody production induced by ovalbumin, which may be attributed to its sustained-release profile. Accordingly, tannic acid is useful as a simple and user-friendly drug delivery system for protein drugs. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Polyvinyl alcohol (PVA) is considered to be an unsuitable carrier for hot-melt extrusion (HME) due to its low processability. In this study, we focused on a lowly hydrolyzed PVA (JR-05, 70.0–74.0% hydrolyzed, L-PVA) to evaluate the potential use of L-PVA as a carrier for HME using the Hoftyzer and Krevelen method and thermodynamic models. These evaluations for drug-polymer systems based on the Flory-Huggins theory predicted the physicochemical properties of the solubility and miscibility between indomethacin (IND) and PVAs. Prior to initiating formulation studies, construction of IND and PVA phase diagrams provided guidance for design process conditions in HME. On the basis of the results of the validation studies, a conventional grade of PVA (JP-05, 87.0–89.0% hydrolyzed) is unlikely to be suitable as a carrier of HME due to its low plasticity, resulting in rapid recrystallization in the evaluation of in vitro dissolution and stability caused by poor miscibility. On the other hand, JR-05 demonstrated high processability, leading to the preparation of miscible extrudate with a high stability and an excellent in vitro dissolution profile due to its unique micelle-forming capability. These findings suggest that L-PVA brings about new carrier options among non-ionic polymers for HME. [ABSTRACT FROM AUTHOR]

In our previous study, we demonstrated that folate-appended methyl‑β‑cyclodextrin (FA-M-β-CyD) was a promising antitumor agent for the treatment of folate receptor-α (FR-α)-expressing tumors. In the present study, to enhance the antitumor effect of FA-M-β-CyD against FR-α- and CD44-expressing colorectal cancer cells, we synthesized a dual targeting supramolecular complex composed of FA-M-β-CyD and adamantane-grafted hyaluronic acid (Ad-HA). The supramolecular complex of Ad-HA/FA-M-β-CyD showed higher cytotoxic activity in HCT116 cells (FR-α (+), CD44 (+)), a human colon cancer cell line, than FA-M-β-CyD alone. In addition, the cytotoxic activity of Ad-HA/FA-M-β-CyD was significantly impaired by the addition of FA and HA, as inhibitors of FR-α and CD44, respectively. Furthermore, tetramethylrhodamine isothiocyanate (TRITC)-labeled FA-M-β-CyD was efficiently internalized into HCT116 cells through supramolecular complexation with Ad-HA, compared to that of TRITC-FA-M-β-CyD alone. Additionally, Ad-HA/FA-M-β-CyD induced mitophagy in HCT116 cells. These results suggest that Ad-HA/FA-M-β-CyD targeted HCT116 cells, as well as induced mitophagy-mediated cell death. Notably, an intravenous injection of the Ad-HA/FA-M-β-CyD complex in a mouse model of colorectal cancer significantly ameliorated the growth of tumor polyps. Collectively, these results suggest that Ad-HA/FA-M-β-CyD has antiproliferation effects in tumors, based on the dual targeting activity. [ABSTRACT FROM AUTHOR]

In this study, in order to investigate the potential of the novel polysaccharide sacran from Aphanothece Sacrum for development of atopic dermatitis (AD), we evaluated the potential of pretreatment with topical sacran to prevent the development of hapten (dinitrofluorobenzene: DNFB)-induced AD-like disease in mice. In the AD model mice, sacran markedly ameliorated AD symptoms such as scratching behavior and edema in ear. In addition, sacran significantly increased water content of the stratum corneum which regulates the skin barrier function. Furthermore, sacran significantly inhibited inflammatory cytokine and chemokine mRNA levels in the dermatitis skin as well as the IgE antibody level in serum. Sacran inhibited inflammatory cytokines mRNA production from Jurkat cells derived from human leukemia T cells after stimulation with phorbol 12-myristate 13-acetate/ionomycin. Meanwhile, sacran did not inhibit the proliferation of primary B cells stimulated with lipopolysaccharide. These results suggest that sacran has good anti-allergic effect on AD model mice, probably due to the amelioration of skin barrier function and suppression of immune responses. [ABSTRACT FROM AUTHOR]

Atopic dermatitis (AD) is a skin disorder characterized by filaggrin (FLG) defect. We evaluated sacran’s effects on dust-mite extracts (DME)-induced AD-like disease and also its effect on profilaggrin (proFLG) in a murine model of 2,4- dinitroflurobenze (DNFB)-induced contact hypersensitivity. In the murine AD-like disease model, allergic NC/Nga mice (N = 60) were randomly divided into five treatment groups of 12 animals each: 0.2% and 1%sacran; 0.1% Tacrolimus ; Vaseline and buffer-treated controls. Blood samples were drawn and serum levels of representative Th-1, Th-2 and also Th-17 (IL-17A) cytokines were assayed by Cytometric Bead Array (CBA). In the contact hypersensitivity model, diseased NC/Nga mice (N = 20) were divided into four groups of five mice each [0.05%sacran, 0.05% chondroitin sulfate (CS), 0.5% prednisolone (PD), non-treated control group] and were treated for 14 days. Skin biopsies were performed for the measurement of proFLG-mRNA by real-time PCR. Sacran solutions and 0.1% Tacrolimus reduced disease severity, suppressed histological changes and decreased the serum Th-1 (IFN-γ, TNF-α, IL-2) and Th-2 (IL-4, IL-6, IL-10) cytokines in allergic mice (vs. controls). Additionally, a marked increase of proFLG-mRNA expression was observed in 0.05%sacran group (vs. control 0.05% CS and 0.5% PD groups). Thus, Sacran might be useful as a natural skin barrier enhancer and anti-allergic agent. [ABSTRACT FROM AUTHOR]

Curcumin is one of promising agents to accelerate the wound-healing process. However, the efficacy of curcumin is limited due to its poor water solubility and stability. To enhance the properties of curcumin, 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CyD) can be used through complexation. Recently, we revealed that sacran has the potential to form a hydrogel film (HGF) as a wound dressing material. Therefore, in the present study, we investigated the wound healing ability of curcumin/HP-γ-CyD (Cur/HP-γ-CyD) complex in sacran-based HGF (Sac-HGF). We prepared the Cur/HP-γ-CyD complex in Sac-HGF without surface roughness. Additionally, the amorphous form in the Cur/HP-γ-CyD complex in Sac-HGF were observed. In contrast, the curcumin in Sac-HGF and curcumin/HP-γ-CyD physical mixture in Sac-HGF formed inhomogeneous films due to crystallization of curcumin. Furthermore, HP-γ-CyD played an important role to increase the elastic modulus of the Sac-HGF with high re-swelling ability. The Cur/HP-γ-CyD complex in Sac-HGF maintained antioxidant properties of curcumin. Curcumin was gradually released from the HP-γ-CyD complex in Sac-HGF. Notably, the Cur/HP-γ-CyD complex in Sac-HGF provided the highest wound healing ability in hairless mice. These results suggest that the Cur/HP-γ-CyD complex in Sac-HGF has the potential for use as a new transdermal therapeutic system to promote the wound-healing process. [ABSTRACT FROM AUTHOR]

A wound dressing is one of the essential approaches for preventing further harm to cutaneous wounds as well as promoting wound healing. Therefore, to achieve ideal wound healing, the development of advanced dressing materials is necessary. Recently, we revealed that a novel megamolecular polysaccharide, sacran, has potential properties as a biomaterial in a physically cross-linked hydrogel film (HGF) for wound dressing application. In this study, to enhance the wound-healing properties of sacran hydrogel film (Sac-HGF) further, we fabricated and characterized novel Sac-HGFs containing cyclodextrins (CyDs). The sacran/α-CyD film (Sac/α-CyD-HGF) and sacran/γ-CyD HGF (Sac/γ-CyD-HGF), but not sacran/β-CyD HGF (Sac/β-CyD-HGF), were well prepared without surface roughness. Powder X-ray diffraction (XRD) patterns of the Sac/γ-CyD-HGFs showed a totally amorphous state compared to that shown by Sac/α-CyD-HGFs. Furthermore, the addition of γ-CyD to Sac-HGFs significantly increased the swelling ratio, porosity, and moisture content of the HGFs, compared to those of the Sac-HGF without CyDs. The Sac/γ-CyD-HGFs were not cytotoxic against NIH3T3 cells, a murine fibroblast cell line. Notably, the Sac/γ-CyD-HGFs significantly improved wound healing in mice, compared to that achieved with the Sac-HGF without γ-CyD. These results suggest that γ-CyD has the potential to promote the wound healing ability of Sac-HGF. [ABSTRACT FROM AUTHOR]

The thin hydrogel films consisting of water-swollen polymer networks can potentially be applied for biomedical fields. Recently, natural polysaccharides have great attentions to be developed as wound healing and protection. In the present study, we newly prepared and characterized a physically crosslinked-hydrogel film composed of a novel megamolecular polysaccharide sacran for wound dressing application. We successfully fabricated a physically crosslinked-sacran hydrogel film by a solvent-casting method. The thickness of a sacran hydrogel film was lower than that of a sodium alginate (Na-alginate) film. Importantly, the swollen ratio of a sacran hydrogel film in water at 24 h was 19-fold, compared to initial weight. Meanwhile, a Na-alginate hydrogel film was completely broken apart after rehydration. Moreover, a sacran hydrogel film did not show any cytotoxicity on NIH3T3 cells, a murine fibroblast cell line. The in vivo skin hydration study revealed that a sacran hydrogel film significantly increased the moisture content on hairless mice skin and considerably improved wound healing ability, compared to control (non-treated), probably due to not only the moisturing effect but also the anti-inflammatory effect of sacran. These results suggest that sacran has the potential properties as a basic biomaterial in a hydrogel film for wound dressing application. [ABSTRACT FROM AUTHOR]

Objectives The development of safe gene transfer carriers with high transfection efficiency, which does not affect the cell function, is a challenging issue. In this study, we examined the effects of α-cyclodextrin (α- CyD)/dendrimer conjugate (α- CDE ( G3)) on nitric oxide ( NO) production in murine macrophages J774.1 cells stimulated with toll-like receptors ( TLR) ligands. Methods NO production from macrophages stimulated with TLR ligands was determined by the Griess method. Transfection efficiency of α- CDE ( G3)/plasmid DNA (p DNA) complex was quantified by a luminometer. Key findings α- CDE ( G3) significantly inhibited NO production from J774.1 cells stimulated with TLR ligands. α- CyD molecules in α- CDE ( G3) had no effect on NO production. The inhibitory effect of α- CDE ( G3) on NO production might be attributed to the dendrimer ( G3). Increasing the degree of substitution ( DS) of α- CyD in the α- CDE ( G3) molecule was accompanied by a significant decrease in the inhibition of NO production. Furthermore, higher gene transfection efficiency of α- CDE ( G3)/p DNA complex was observed upon increasing the DS of α- CyD. Conclusions α- CDE ( G3) with high DS value of α- CyD may be considered as a safe gene transfer carrier that does not adversely affect NO production from macrophages stimulated with TLR ligands. [ABSTRACT FROM AUTHOR]

Objectives GM1-gangliosidosis is an inherited disorder characterized by the accumulation of GM1-gangliosides in many tissues and organs, particularly in the brain. Currently, there is no treatment available for patients with ganglioside storage diseases. Therefore, we investigated the effects of cyclodextrins ( Cy Ds) on the GM1-ganglioside level in EA1 cells, fibroblasts from patients with GM1-gangliosidosis. Methods The concentrations of cholesterol and phospholipids in supernatants were determined by Cholesterol E-test Wako and Phospholipid C-test Wako, respectively. The effects of Cy Ds on GM1-ganglioside levels in EA1 cells using fluorescence-labelled cholera toxin B-subunit, which can bind to GM1-gangliosides specifically, were investigated by flow cytometry and confocal laser scanning microscopy. Key findings The treatment with methylated Cy Ds, hydroxypropylated Cy Ds and branched Cy Ds decreased GM1-ganglioside levels in EA1 cells at 1 m m for 24 h. Unexpectedly, there was no significant change in the efflux of cholesterol or phospholipids from the cells after treatment with Cy Ds under the same experimental conditions, indicating that the efflux of membrane components is not associated with down-regulation of GM1-ganglioside levels in EA1 cells upon Cy Ds treatment. Conclusions Cy Ds may have the potential as drugs for GM1-gangliosidosis, although the mechanism should be thereafter clarified. [ABSTRACT FROM AUTHOR]

In a recent study, we attempted to confer a tumor-selective cytotoxic activity to methyl-β-cyclodextrin (M-β-CyD), we synthesized folate-conjugated M-β-CyD (FA-M-β-CyD), and demonstrated the potential of FA-M-β-CyD as a novel anticancer agent at a high dose. In the present study, to expand the application of FA-M-β-CyD for cancer chemotherapy, we evaluated the potential of FA-M-β-CyD as a tumor-targeting anticancer drug carrier at a low dose. FA-M-β-CyD formed an inclusion complex with doxorubicin (DOX) with a high-stability constant (3.0 × 105 M−1). Antitumor activity of DOX was increased by the complexation with FA-M-β-CyD, but not with folate-conjugated β-CyD (FA-β-CyD) or M-β-CyD in KB cells, a folate receptor-α (FR-α)-expressing cell line. Also, FA-M-β-CyD increased antitumor activity of paclitaxel, a class IV compound in the biopharmaceutical classification system (BCS), but not 5-fluorouracil, a class III compound in the BCS. Furthermore, FA-M-β-CyD enhanced cellular uptake of DOX through a complexation in KB cells (FR-α (+)), compared to FA-β-CyD and M-β-CyD. The DOX/FA-M-β-CyD complex showed markedly high antitumor activity, compared to DOX alone and DOX/M-β-CyD complex, after an intravenous administration to FR-α-expressing tumor cell-bearing mice. These findings suggest that FA-M-β-CyD could be useful as a tumor-selective carrier for anticancer drugs. [ABSTRACT FROM AUTHOR]

We reported that per-6-folic acid (FA)-appendedβ-cyclodextrin (β-CyD) possessing two caproic acids betweenFA and a β-CyD molecule as a spacer (Fol-c2-β-CyD)could be useful as a promising antitumor drug carrier. However, theeffects of the cavity size and the spacer length on the carrier abilityare not still known. In this study, we designed and evaluated theFA-appended three kinds of CyDs possessing a caproic acid as a spacerbetween FA and a CyD molecule (Fol-c1-CyDs) as a tumortargeting carrier for antitumor drugs. The stability constant of theFol-c1-β-CyD/doxorubicin (DOX) complex was much higherthan those of Fol-c1-α-CyD and Fol-c1-γ-CyDat pH 7.3. Antitumor activity of DOX was increased by the complexationwith Fol-c1-β-CyD, but not with Fol-c1-α-CyD or Fol-c1-γ-CyD in KB cells, a folatereceptor-α-positive cell line. Also, Fol-c1-β-CyDincreased antitumor activities of paclitaxel and vinblastine, butnot 5-fluorouracil. Furthermore, Fol-c1-β-CyD acceleratedcellular uptake of DOX and inhibited its efflux from KB cells. TheFol-c1-β-CyD/DOX complex showed much higher antitumoractivity than DOX alone after intratumoral and intravenous administrationsto tumor-bearing mice with a negligible change of the blood chemistryvalues. These findings suggest that Fol-c1-β-CyDcould be useful as a tumor-selective carrier for antitumor drugs. [ABSTRACT FROM AUTHOR]

Abstract: Methyl-β-cyclodextrin (M-β-CyD), which is widely used as a lipid rafts disrupting agent, is known to induce cytotoxicity at high concentration. In the present study, we investigated the potential of M-β-CyD as an antitumor drug. M-β-CyD markedly caused apoptotic cell-death in KB cells, a human oral squamous carcinoma cell line, Ihara cells, a highly pigmented human melanoma cell line, and M213 cells, a human cholangiocarcinoma cell line, through cholesterol depletion in cell membranes. The DNA content and mitochondrial transmembrane potential in KB cells were significantly decreased after treatment with M-β-CyD. Additionally, M-β-CyD elevated the caspase-3/7 activity in KB cells. Meanwhile, M-β-CyD did not induce the formation of autophagic vacuoles in KB cells. M-β-CyD drastically inhibited the tumor growth after intratumoral injection to Colon-26 cells-bearing mice. These results strongly suggest that M-β-CyD induced apoptosis in tumor cells and had the potential a novel antitumor agent and/or its lead compound. [Copyright &y& Elsevier]

Abstract: The widespread use of various cyclodextrin (CyD)-appended polymers and polyrotaxanes as gene carriers has been reported. Among the various polyamidoamine dendrimer (dendrimer) conjugates with CyDs (CDE), the dendrimer (G3) conjugate with α-CyD having an average degree of substitution (DS) of 2.4 (α-CDE (G3, DS 2)) displayed remarkable properties as DNA carriers. In an attempt to develop cell-specific gene transfer carriers, we prepared some sugar-appended α-CDEs, e.g. mannosylated, galactosylated, and lactosylated α-CDEs. In addition, PEGylated Lac-α-CDEs (G3) were prepared and evaluated as a hepatocyte-selective and serum-resistant gene transfer carrier. Moreover, PEGylated-α-CDE/CyD polypseudorotaxane systems for novel sustained DNA release system have been developed. Interestingly, glucronylglucosyl-β-cyclodextrin (GUG-β-CyD) conjugates with dendrimer (G2) (GUG-β-CDE (G2)) had superior gene transfer activity to α-CDE (G2), expecting a development of new series of sugar-appended CDEs over α-CDEs (G2). Collectively, sugar-appended α-CDEs have the potential as novel cell-specific and safe carriers for DNA. [Copyright &y& Elsevier]

Gene, short hairpin RNA (shRNA) and small interfering RNA (siRNA) delivery can be particularly used for the treatment of diseases by the entry of genetic materials mammalian cells either to express new proteins or to suppress the expression of proteins, respectively. Polyamidoamine (PAMAM) Starburst™ dendrimers are used as non-viral vectors (carriers) for gene, shRNA and siRNA delivery. Recently, multifunctional PAMAM dendrimers can be used for the wide range of biomedical applications including intracellular delivery of genes and nucleic acid drugs. In this context, this review paper provides the recent findings on PAMAM dendrimer conjugates with cyclodextrins (CyDs) for gene, shRNA and siRNA delivery. [ABSTRACT FROM AUTHOR]

Cyclodextrin (CyD)-based nanoparticles and polyamidoamine (PAMAM) starburst dendrimers (dendrimers) are used as novel carriers for DNA and RNA. Recently, small interfering RNA (siRNA) complex with β-CyD-containing polycations (CDP) having adamantine-PEG or adamantine-PEG-transferrin underwent a phase I study for treatment of solid tumors. Multifunctional dendrimers can be used for a wide range of biomedical applications, including the interaction and intracellular delivery of DNA and RNA. The present review will address the latest developments in dendrimer conjugates with cyclodextrins for siRNA delivery including the novel sustained release system. [ABSTRACT FROM AUTHOR]

Abstract: Nonviral gene delivery suffers from a number of limitations including short transgene expression times and low transfection efficiency. In this study, we examined whether polypseudorotaxanes (PPRXs) of polyethylene glycol (PEG, molecular weight: 2,000)-grafted α-cyclodextrin (α-CyD)/polyamidoamine dendrimer conjugate (PEG-α-CDE) with CyDs have the potential for the novel sustained release systems for plasmid DNA (pDNA). The PEG-α-CDE/pDNA complex formed PPRXs with α-CyD and γ-CyD solutions, but not with β-CyD solution. In the PEG-α-CDE/CyDs PPRX systems, 20.6mol of α-CyD and 11.8mol of γ-CyD were involved in the PPRXs formation with one PEG chain by α-CyD and γ-CyD, respectively, consistent with in the PEG-dendrimer/CyDs systems. PEG-α-CDE/pDNA/α-CyD PPRX and PEG-α-CDE/pDNA/γ-CyD PPRX formed hexagonal and tetragonal columnar channels in the crystalline phase, respectively. In addition, the CyDs PPRX provided the sustained release of pDNA from PEG-α-CDE complex with pDNA at least 72h in vitro. The release of pDNA from CyDs PPRX retarded as the volume of dissolution medium decreased. Furthermore, the PEG-α-CDE/γ-CyD PPRX system showed sustained transfection efficiency after intramuscular injection to mice at least for 14days. These results suggest that the PEG-α-CDE/CyD PPRX systems are useful for novel sustained DNA release systems. [Copyright &y& Elsevier]

The purpose of this study is to evaluate in vitro gene delivery mediated by asialofetuin-appended cationic liposomes (AFliposomes) associating cyclodextrins (CyD/AF-liposomes) as a hepatocyte-selective nonviral vector. Of various CyDs, AFliposomes 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. [ABSTRACT FROM AUTHOR]

Abstract: Topical application of nuclear factor-κB (NF-κB) decoy appears to provide a novel therapeutic potency in the treatment of inflammation and atopic dermatitis. However, it is difficult to deliver NF-κB decoy oligonucleotides (ODN) into the skin by conventional methods based on passive diffusion because of its hydrophilicity and high molecular weight. In this study, we evaluated the in vitro transdermal delivery of fluorescein isothiocyanate (FITC)-NF-κB decoy ODN using a pulse depolarization (PDP) iontophoresis. In vitro iontophoretic experiments were performed on isolated C57BL/6 mice skin using a horizontal diffusion cell. The apparent flux values of FITC-NF-κB decoy ODN were enhanced with increasing the current density and NF-κB decoy ODN concentration by iontophoresis. Accumulation of FITC-NF-κB decoy ODN was observed at the epidermis and upper dermis by iontophoresis. In mouse model of skin inflammation, iontophoretic delivery of NF-κB decoy ODN significantly reduced the increase in ear thickness caused by phorbol ester as well as the protein and mRNA expression levels of tumor necrosis factor-α (TNF-α) in the mice ears. These results suggest that iontophoresis is a useful and promising enhancement technique for transdermal delivery of NF-κB decoy ODN. [Copyright &y& Elsevier]

Abstract: Cyclodextrins (CyDs), which are widely used to increase the solubility of drug in pharmaceutical fields, are known to induce hemolysis and cytotoxicity at high concentrations. However, it is still not unclear whether cell death induced by CyDs is apoptosis or not. Therefore, in the present study, we investigated the effects of various kinds of CyDs on apoptosis in the cells such as NR8383 cells, A549 cells and Jurkat cells. Of various CyDs, methylated CyDs inducted cell death under the present experimental conditions, but hydroxypropylated CyDs or sulfobutyl ether-β-CyD (SBE7-β-CyD) did not. Of methylated CyDs, 2,6-di-O-methyl-β-cyclodextrin (DM-β-CyD) and 2,3,6-tri-O-methyl-β-cyclodextrin (TM-β-CyD) markedly caused apoptosis in NR8383 cells, A549 cells and Jurkat cells, through cholesterol depletion in cell membranes. In sharp contrast, 2,6-di-O-methyl-α-cyclodextrin (DM-α-CyD) and methyl-β-cyclodextrin (M-β-CyD) induced cell death in an anti-apoptotic mechanism. DM-β-CyD induced apoptosis through the inhibition of the activation of PI3K-Akt-Bad pathway. Neither p38 MAP kinase nor p53 was contributed to the induction of apoptosis by DM-β-CyD. Additionally, DM-β-CyD significantly decreased mitochondrial transmembrane potential, and then caused the release of cytochrome c from mitochondria to cytosol in NR8383 cells. Furthermore, we confirmed that down-regulation of pro-caspase-3 and activation of caspase-3 after incubation with DM-β-CyD. These results suggest that of methylated CyDs, DM-β-CyD, not DM-α-CyD, induces apoptosis through the PI3K-Akt-Bad pathway, resulting from cholesterol depletion in lipid rafts of cell membranes. [Copyright &y& Elsevier]

We have evaluated the potential use of various polyamidoamine (PAMAM) dendrimer [dendrimer, generation (G) 2-4] conjugates with cyclodextrins (CyDs) as novel DNA and RNA carriers. Among the various dendrimer conjugates with CyDs, the dendrimer (G3) conjugate with α-CyD having an average degree of substitution (DS) of 2.4 [α-CDE (G3, DS2)] displayed remarkable properties as DNA, shRNA and siRNA delivery carriers through the sensor function of α-CDEs toward nucleic acid drugs, cell surface and endosomal membranes. In an attempt to develop cell-specific gene transfer carriers, we prepared sugar-appended α-CDEs. Of the various sugar-appended α-CDEs prepared, galactose- or mannose-appended α-CDEs provided superior gene transfer activity to α-CDE in various cells, but not cell-specific gene delivery ability. However, lactose-appended α-CDE [Lac-α-CDE (G2)] was found to possess asialoglycoprotein receptor (AgpR)-mediated hepatocyte-selective gene transfer activity, both in vitro and in vivo. Most recently, we prepared folate-poly(ethylene glycol)-appended α-CDE [Fol-PαC (G3)] and revealed that Fol-P?C (G3) imparted folate receptor (FR)-mediated cancer cellselective gene transfer activity. Consequently, α-CDEs bearing integrated, multifunctional molecules may possess the potential to be novel carriers for DNA, shRNA and siRNA. [ABSTRACT FROM AUTHOR]

Abstract: In the present study, we examined the effects of α-cyclodextrin (α-CyD), 2-hydroxypropyl-α-cyclodextrin (HP-α-CyD) and 2,6-di-O-methyl-α-cyclodextrin (DM-α-CyD) on the nitric oxide (NO) and interferon-β (IFN-β) production in murine and human macrophages stimulated with Poly I:C and CpG-DNA, toll-like receptor 3 (TLR3) and TLR9 ligands, respectively. DM-α-CyD significantly inhibited NO production in RAW264.7 cells and U937 cells differentiated by phorbol myristate acetate (PMA), murine and human macrophage-like cell lines, respectively, stimulated with Poly I:C without cytotoxicity, but neither α-CyD nor HP-α-CyD did. Meanwhile, the three α-CyDs did not inhibit NO production in RAW264.7 cells stimulated with CpG-DNA. DM-α-CyD inhibited inducible NO synthase (iNOS) and IFN-β expression upon stimulation with Poly I:C. Furthermore, DM-α-CyD markedly decreased the cellular uptake of Poly I:C in RAW264.7 cells. Therefore, DM-α-CyD may be useful as a potent inhibitor for excess activation of macrophages stimulated with Poly I:C. [Copyright &y& Elsevier]

Abstract: The effects of 2,6-di-O-methyl-α-cyclodextrin (DM-α-CyD) on hemolysis and morphological changes in rabbit''s red blood cells (RBC) were examined, compared with those of α-cyclodextrin (CyD) and 2-hydoxypropyl-α-cyclodextrin (HP-α-CyD). The hemolytic activity of α-CyDs increased in the order of HP-α-CyD<α-CyD

Abstract: The potential use of hydrophilic cyclodextrins (CyDs) as an inhibitor for lipopolysaccharide (LPS) was examined. Of the five CyDs used in this study, dimethylacetyl-β-cyclodextrin (DMA7-β-CyD) had greater inhibitory activity than other CyDs against the production of nitric oxide (NO) and various proinflammatory cytokines including tumor necrosis factor-α (TNF-α) in murine macrophages stimulated with two serotypes of LPS and lipid A. The inhibitory effect of DMA7-β-CyD on NO production was also observed in macrophages stimulated with lipoteichoic acid (LTA), but not peptidoglycan (PGN), polyinosinic–polycytidylic acid (poly I:C) or CpG oligonucleotide (CpG-ODN). Several studies have suggested that the inhibitory effects of DMA7-β-CyD could be ascribed to the interaction with LPS. Simultaneous administration of DMA7-β-CyD not only intraperitoneally but also intravenously and intraperitoneal injection of aqueous solution containing LPS and d-galactosamine in murine endotoxin shock model suppressed fatality. Also, DMA7-β-CyD decreased blood level of TNF-α as well as serum levels of aspartate transaminase (AST) and alanine transaminase (ALT) in mice. In conclusion, DMA7-β-CyD may have promise as a new therapeutic agent for endotoxin shock induced by LPS. [Copyright &y& Elsevier]

Abstract: The potential use of α-cyclodextrin and its hydrophilic α-cyclodextrin derivatives (α-CyDs) as antagonists against lipopolysaccharide (LPS), which stimulates the nitric oxide (NO) and tumor necrosis factor-α (TNF-α) production as well as nuclear factor-κB (NF-κB) activation in macrophages was examined. Of three α-CyDs used in the present study, 2,6-di-O-methyl-α-CyD (DM-α-CyD) had greater inhibitory activity than did the other CyDs against NO and TNF-α production through an impairment of gene expression in macrophage cell lines and primary macrophages stimulated with LPS and lipid A in a concentration-dependent manner. Concomitantly, DM-α-CyD inhibited NF-κB translocation into nucleus. These inhibitory effects of DM-α-CyD could be attributed to the release of CD14 from lipid rafts caused by an efflux of phospholipids, but not cholesterol. These results suggest that DM-α-CyD may have promise as a potent and unique antagonist for excess activation of macrophages stimulated with LPS. [Copyright &y& Elsevier]

• Few stabilizing agents stabilize antibody against both thermal and shaking stresses. • PpRX hydrogels stabilized antibody against both thermal and shaking stresses. • The stabilizing effects of the PpRX hydrogels were greater than common gels. • Components of the PpRX hydrogels are biocompatible and low cost. • PpRX hydrogels were prepared by only mixing CyD and pluronics. Recently, antibody drugs have been used worldwide, and based on worldwide sales, 7 of the top 10 pharmaceutical products in 2019 were antibody-based drugs. However, antibody drugs often form aggregates upon thermal and shaking stresses with few efficient stabilizing agents against both stresses. Herein, we developed polypseudorotaxane (PpRX) hydrogels consisting of cyclodextrins (CyDs) and polyethylene glycol (PEG)-polypropylene glycol (PPG)-PEG block copolymers (Pluronics F108, F87, F68, and L44), and evaluated their utility as antibody stabilizing agents. α- and γ-CyDs formed PpRX hydrogels with Pluronics, where CyD/F108 gels showed remarkable stabilizing effects for human immunoglobulin G (IgG) against both thermal and shaking stresses beyond CyD/PEG gels or generic gels. The effects were probably due to the interaction between IgG and the free PPG block of Pluronic F108, resulting in the strong IgG retention in the gels. These findings suggest the great potential of CyD/Pluronic gels as pharmaceutical materials for antibody formulations. [ABSTRACT FROM AUTHOR]

Cyclodextrins (CDs) are essential in the pharmaceutical industry and have long been used as food and pharmaceutical additives. CD-based interlocked molecules, such as rotaxanes, polyrotaxanes, catenanes, and polycatenanes, have been synthesized and have attracted considerable attention in supramolecular chemistry. Among them, CD polyrotaxanes have been employed as slide-ring materials and biomaterials. CD polycatenanes are new materials; therefore, to date, no examples of applied research on CD polycatenanes have been reported. Consequently, we expect that applied research on CD polycatenanes will accelerate in the future. This review article summarizes the syntheses and structural analyses of CD polyrotaxanes and polycatenanes to facilitate their applications in the pharmaceutical industry. We believe that this review will promote further research on CD-based interlocked molecules. [Display omitted] [ABSTRACT FROM AUTHOR]

Niemann-Pick type C disease (NPC) is a fatal, autosomal recessive disorder, which causes excessive accumulation of free cholesterol in endolysosomes, resulting in progressive hepatomegaly and neurodegeneration. Currently, 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) is used at a high dose for the treatment of NPC, risking lung toxicity and hearing loss during treatment. One method to reduce the required dose of HP-β-CyD for the treatment of hepatomegaly is to actively deliver β-cyclodextrin (β-CyD) to hepatocytes. Previously, we synthesized lactosyl-β-CyD (Lac-β-CyD) and demonstrated that it lowers cholesterol in NPC model liver cells. In the present study, we studied the efficacy and safety of Lac-β-CyD treatment of hepatomegaly in Npc1−/− mice. After subcutaneous administration, Lac-β-CyD accumulated in the liver and reduced hepatomegaly with greater efficacy than HP-β-CyD. In addition, subcutaneous administration of a very high dose of Lac-β-CyD was less toxic to the lungs than HP-β-CyD. Notably, the accumulation of intracellular free cholesterol in endolysosomes of NPC-like liver cells was significantly lower after administration of Lac-β-CyD than after treatment with HP-β-CyD. In conclusion, these results suggest that Lac-β-CyD is a candidate for the effective treatment of hepatomegaly in NPC. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is a heterogenous myeloid neoplasm that remains challenging to treat. Because intensive conventional chemotherapy reduces survival rates in elderly patients, drugs with lower toxicity and fewer side effects are needed urgently. 2-Hydroxypropyl-β-cyclodextrin (HP-β-CyD) is used clinically as a pharmaceutical excipient for poorly water-soluble drugs. Previously, we showed that HP-β-CyD exerts antitumor activity by disrupting cholesterol homeostasis. Recently, we developed folate-conjugated HP-β-CyD (FA-HP-β-CyD) and demonstrated its potential as a new antitumor agent that induces not only apoptosis, but also autophagic cell death; however, we do not know whether FA-HP-β-CyD exerts these effects against AML. Here, we investigated the effects of FA-HP-β-CyD on folate receptor (FR)-expressing AML cells. We found that the cytotoxic activity of FA-HP-β-CyD against AML cells was stronger than that of HP-β-CyD. Also, FA-HP-CyD induced the formation of autophagosomes in AML cell lines. FA-HP-β-CyD increased the inhibitory effects of cytarabine and a BCL-2-selective inhibitor, Venetoclax, which are commonly used treat elderly AML patients. Notably, FA-HP-β-CyD suppressed the proliferation of AML cells in BALB/c nude recombinase-activating gene-2 (Rag-2)/Janus kinase 3 (Jak3) double-deficient mice with AML. These results suggest that FA-HP-β-CyD acts as a potent anticancer agent for AML chemotherapy by regulating autophagy. [ABSTRACT FROM AUTHOR]

Background: Niemann–Pick disease type C (NPC) is a fatal neurodegenerative disorder caused by abnormal intracellular cholesterol trafficking. Cyclodextrins (CDs), the most promising therapeutic candidates for NPC, but with concerns about ototoxicity, are cyclic oligosaccharides with dual functions of unesterified cholesterol (UC) shuttle and sink that catalytically enhance the bidirectional flux and net efflux of UC, respectively, between the cell membrane and the extracellular acceptors. However, the properties of CDs that regulate these functions and how they could be used to improve treatments for NPC are unclear. Methods: We estimated CD–UC complexation for nine CD derivatives derived from native α‐, β‐, and γ‐CD with different cavity sizes, using solubility and molecular docking analyses. The stoichiometry and complexation ability of the resulting complexes were investigated in relation to the therapeutic effectiveness and toxicity of each CD derivative in NPC experimental models. Findings: We found that shuttle and sink activities of CDs are dependent on cavity size‐dependent stoichiometry and substituent‐associated stability of CD–UC complexation. The ability of CD derivatives to form 1:1 and 2:1 complexes with UC were correlated with their ability to normalize intracellular cholesterol trafficking serving as shuttle and with their cytotoxicity associated with cellular UC efflux acting as sink, respectively, in NPC model cells. Notably, the ability of CD derivatives to form an inclusion complex with UC was responsible for not only efficacy but ototoxicity, while a representative derivative without this ability negligibly affected auditory function, underscoring its preventability. Conclusions: Our findings highlight the importance of strategies for optimizing the molecular structure of CDs to overcome this functional dilemma in the treatment of NPC. [ABSTRACT FROM AUTHOR]

In this study, we newly developed the ternary complexes consisting of lactosylated dendrimer (generation 3)/α-cyclodextrin conjugate (Lac-α-CDE), siRNA and the anionic polysaccharide sacrans, and evaluated their utility as siRNA transfer carriers. Three kinds of the low-molecular-weight sacrans, i.e. sacran (100) (Mw 44,889 Da), sacran (1000) (Mw 943,692 Da) and sacran (10,000) (Mw 1,488,281 Da) were used. Lac-α-CDE/siRNA/sacran ternary complexes were prepared by adding the low-molecular-weight sacrans to the Lac-α-CDE/siRNA binary complex solution. Cellular uptake of the ternary complex with sacran (100) was higher than that of the binary complex or the other ternary complexes with sacran (1000) and sacran (10,000) in HepG2 cells. Additionally, the ternary complex possessed high serum resistance and endosomal escaping ability in HepG2 cells. High liver levels of siRNA and Lac-α-CDE were observed after the intravenous administration of the ternary complex rather than that of the binary complex. Moreover, intravenous administration of the ternary complex (siRNA 5 mg/kg) induced the significant RNAi effect in the liver of mice with negligible change of blood chemistry values. Therefore, a ternary complexation of the Lac-α-CDE/siRNA binary complex with sacran is useful as a hepatocyte-specific siRNA delivery system. [ABSTRACT FROM AUTHOR]

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 44,889 Da, 943,692 Da and 1,488,281 Da, 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. [ABSTRACT FROM AUTHOR]

Sustained and controllable release of insulin is strongly required to achieve the ideal treatment of diabetes. We previously developed “self-assembly PEGylation retaining activity (SPRA) technology” via a host–guest interaction between PEGylated β-cyclodextrin and adamantane-appended insulin, and resulting PEGylated insulin was termed SPRA-insulin. So far, we also demonstrated that covalently PEGylated insulin forms polypseudorotaxanes (PPRXs) with cyclodextrins (PPRX technology). In the present study, we designed and evaluated the combination system of SPRA technology and PPRX technology to achieve a sustained and controllable release system of insulin. SPRA-insulin formed PPRXs with α-cyclodextrin and γ-cyclodextrin. In addition, SPRA-insulin/cyclodextrin PPRXs provided sustained and controllable release of insulin beyond the each single technology both in vitro and in vivo . These results suggest that the combination system of SPRA technology and PPRX technology is useful for design of a sustained and controllable release system of insulin. [ABSTRACT FROM AUTHOR]

[Display omitted] Recent viral pandemics have increased global demand for vaccines. However, the supply of effective and safe vaccine not only to developed countries but also developing countries with inadequate storage equipment is still challenging due to the lack of robust systems which improve the efficacy and the stability of vaccines with few side effects. In our previous study, polypseudorotaxane (PPRX) hydrogels based on cyclodextrin (CyD) and polyethylene glycol (PEG) significantly improved the stability of antibody preparations and showed no serious adverse effects after subcutaneous injection, suggesting the possibility as safe vaccine formulations to stabilize an antigen protein. Moreover, recent studies have reported that one of the CyD derivatives, hydroxypropyl-β-CyD (HP-β-CyD), acts as an adjuvant to enhance protective type-2 immune responses. However, it is still unknown that CyD PPRX hydrogels enhance not only the stability of an antigen protein but also its immunogenicity with tolerable side effects. Here, we demonstrate that α- and γ-CyD PPRX hydrogels containing an antigen protein significantly induce antigen-specific type-2 immune responses. Moreover, α- and γ-CyD PPRX hydrogels showed negligible local irritation at the injection site, although subcutaneous injection of α-CyD alone induced skin lesion. Finally, shaking stability of the antigen protein at room temperature was significantly improved by being included in α- and γ-CyD PPRX hydrogels. These results propose the possibility of α- and γ-CyD PPRX hydrogels as novel vaccine formulations which improve both the immunogenicity and stability of an antigen protein with suppressed local irritation. [ABSTRACT FROM AUTHOR]

2-Hydroxypropyl-β-cyclodextrin (HP-β-CyD) is a cyclic oligosaccharide that is widely used as an enabling excipient in pharmaceutical formulations, but also as a cholesterol modifier. HP-β-CyD has recently been approved for the treatment of Niemann-Pick Type C disease, a lysosomal lipid storage disorder, and is used in clinical practice. Since cholesterol accumulation and/or dysregulated cholesterol metabolism has been described in various malignancies, including leukemia, we hypothesized that HP-β-CyD itself might have anticancer effects. This study provides evidence that HP-β-CyD inhibits leukemic cell proliferation at physiologically available doses. First, we identified the potency of HP-β-CyD in vitro against various leukemic cell lines derived from acute myeloid leukemia (AML), acute lymphoblastic leukemia and chronic myeloid leukemia (CML). HP-β-CyD treatment reduced intracellular cholesterol resulting in significant leukemic cell growth inhibition through G2/M cell-cycle arrest and apoptosis. Intraperitoneal injection of HP-β-CyD significantly improved survival in leukemia mouse models. Importantly, HP-β-CyD also showed anticancer effects against CML cells expressing a T315I BCR-ABL mutation (that confers resistance to most ABL tyrosine kinase inhibitors), and hypoxia-adapted CML cells that have characteristics of leukemic stem cells. In addition, colony forming ability of human primary AML and CML cells was inhibited by HP-β-CyD. Systemic administration of HP-β-CyD to mice had no significant adverse effects. These data suggest that HP-β-CyD is a promising anticancer agent regardless of disease or cellular characteristics. [ABSTRACT FROM AUTHOR]

The article focuses on nusinersen improved respiratory function in spinal muscular atrophy (SMA) type 2. Topics include the SMA is a neuromuscular disorder has characterized by progressive motor paralysis and respiratory insufficiency, the respiratory insufficiency is fatal in SMA type 1 and a serious problem for SMA types 2 and 3, and the retrospective study in Japan has showed that noninvasive positive pressure ventilation (NPPV) was needed for 52% and 13% of SMA types 2 and 3 patients.

Pharmaceutical excipients, such as surfactants, amino acids, and polymers, have often been used to improve the physicochemical properties of protein drugs. However, the effects of these additives are limited because of factors such as their weak interactions with protein drugs. In the present study, we evaluated the application of a supramolecular polymer, aminated polyrotaxane (NH 2 -PRX), which can strongly interact with protein drugs via its dynamic and transformable properties, as a new pharmaceutical excipient for these agents. As a conventional control polymer with low mobility and average complexation ability, aminated dextran (NH 2 -DEX) was also prepared. NH 2 -PRX significantly reduced the aggregation of antibodies induced by shaking, compared with NH 2 -DEX. The adsorption of insulin onto glass and polypropylene containers was also reduced by the addition of NH 2 -PRX. In addition, the in vivo bioactivity of insulin was completely retained in the presence of NH 2 -PRX. Moreover, severe adverse effects were not observed following the administration of NH 2 -PRX. These findings indicate the potential use of NH 2 -PRX as a transformable pharmaceutical excipient for protein drugs. [ABSTRACT FROM AUTHOR]

The oral bioavailability of drugs is limited by factors such as poor membrane permeability, low solubility, and low dissolution rate. Silymarin (SLM) is a health-food active ingredient that is good for immunosuppression and tumor suppression. However, obtaining a good oral bioavailability is difficult owing to its poor solubility and low dissolution ability. To overcome these concerns, we previously prepared SLM nanoparticles (NPs) using the high-pressure crystallization method (PureNanoTM) and freeze-dried them with erythritol (Ery) or hydroxypropyl-β-CyD (HP-β-CyD) as a water-soluble dispersion stabilizer. In the present study, we investigated the mechanism underlying the improved absorption of SLM/hypromellose (HPMC)/HP-β-CyD NPs after oral administration. The SLM/HPMC nano-suspension prepared using PureNanoTM exhibited a narrow size distribution. The size of the SLM/HPMC/HP-β-CyD NPs was approximately 250 nm after hydration. The SLM/HPMC/HP-β-CyD NPs were rapidly dissolved, and demonstrated a high solubility under supersaturated conditions. Additionally, they exhibited good wettability and their membrane permeability was improved compared with that of SLM original powder. These results suggest that the formulation of SLM NPs using PureNanoTM and freeze-drying with HP-β-CyD improves the absorption of SLM after oral administration by enhancing solubility, wettability, and membrane permeability. [ABSTRACT FROM AUTHOR]

Niemann–Pick disease type C (NPC) is a lysosomal lipid storage disorder characterized by progressive neurodegeneration and hepatic dysfunction. A cyclic heptasaccharide, 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), is currently under clinical investigation for NPC, but its adverse events remain problematic. We previously identified that a cyclic octasaccharide, 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD), also ameliorated NPC manifestations with higher biocompatibility than HP-β-CD. However, preclinical studies describing the associations between the biodistribution and pharmacodynamics of these compounds, which are essential for clinical application, are still lacking. Here, we investigated these properties of HP-γ-CD by measuring its organ biodistribution and therapeutic effect after systemic and central administration. The effect of HP-γ-CD on disturbed cholesterol homeostasis appeared within several hours after exposure and persisted for several days in NPC model cells and mice. Tissue distribution indicated that only a small fraction of subcutaneously administered HP-γ-CD rapidly distributed to peripheral organs and contributed to disease amelioration. We found that a subcutaneous dose of HP-γ-CD negligibly ameliorated neurological characteristics because it has limited penetration of the blood–brain barrier; however, an intracerebroventricular microdose unexpectedly attenuated hepatic dysfunction without the detection of HP-γ-CD in the liver. These results demonstrate that central administration of HP-γ-CD can indirectly attenuate peripheral manifestations of NPC. [ABSTRACT FROM AUTHOR]

To develop a novel hepatocyte-selective gene carrier, we prepared polyamidoamine starburst dendrimer (generation 3, G3) conjugates with three functional molecules, i.e. α-cyclodextrin, polyethylene glycol (PEG, molecular weight = 2170) and lactose (PEG-LαCs), and evaluated gene delivery efficiency of these conjugates in vitro and in vivo. PEG-LαC (G3, degrees of substitution of the PEG moiety (DSP) 2.1) showed higher gene transfer activity than other PEG-LαCs (G3, DSP4.0, 6.2) in HepG2 cells, expressing asialoglycoprotein receptor, and the activity decreased in HeLa cells, non-expressing the receptor and in the presence of asialofetuin. High gene transfer activity of PEG-LαC (G3, DSP2.1) was retained even in the presence of 50% serum, although the activity of α-cyclodextrin/lactosylated dendrimer (G3) conjugate (Lac-α-CDE (G3)), which is lacking a PEG moiety, was severely decreased in the presence of 20% serum. PEG-LαC (G3, DSP2.1) provided negligible cytotoxicity up to a charge ratio of 50 (carrier/pDNA) in HepG2 cells and less acute organ toxicity. PEG-LαC (G3, DSP2.1) showed selective gene transfer activity to hepatic parenchymal cells rather than hepatic non-parenchymal cells. These results suggest that PEG-LαC (G3, DSP2.1) is useful as a hepatocyte-selective gene carrier in vitro and in vivo. [ABSTRACT FROM AUTHOR]

In this study, to clarify the utility of polypseudorotaxane (PPRX) hydrogels composed of poly(ethylene glycol) (PEG) and α- or γ-cyclodextrin (α- or γ-CyD) as a sustained-release system for protein drugs, we prepared CyD PPRX hydrogels including lysozyme, and then the release profiles of lysozyme from these hydrogels and the release mechanisms were investigated. The α- and γ-CyD formed PPRX hydrogels by threading onto one PEG chain and two PEG chains, respectively. The formation of α- and γ-CyD PPRX hydrogels including lysozyme was based on physical cross-linking arisen from their columnar structures. The in vitro release rates of lysozyme were markedly decreased by the encapsulation into CyD PPRX hydrogels. In addition, when release data were plotted according to Korsmeyer-Peppas model, the exponent values ( n) in the α- and γ-CyD systems had no statistically significant difference, suggesting that these release mechanisms were almost same. In conclusion, these results suggest that α- and γ-CyD PPRX hydrogels possess the potential as a sustained-release system for lysozyme. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:2891-2899, 2012 [ABSTRACT FROM AUTHOR]

Abstract: We previously reported that glucuronylglucosyl-β-cyclodextrin (GUG-β-CyD) conjugate with polyamidoamine starburst dendrimer (GUG-β-CDE conjugate) with the average degree of substitution (DS) of cyclodextrin (CyD) of 1.8 (GUG-β-CDE conjugate (DS 1.8)), showed remarkably higher gene transfer activity than α-CyD/dendrimer conjugate (α-CDE conjugate (DS 1.2)) and β-CyD/dendrimer conjugate (β-CDE conjugate (DS 1.3)) in vitro and in vivo. In this study, to clarify the enhancing mechanism for high gene transfer activity of GUG-β-CDE conjugate (DS 1.8), we investigated the physicochemical properties, cellular uptake, endosomal escape and nuclear translocation of the plasmid DNA (pDNA) complexes as well as pDNA release from the complexes. The particle size, ζ-potential and cellular uptake of GUG-β-CDE conjugate (DS 1.8)/pDNA complex were mostly comparable to those of α-CDE conjugate (DS 1.2) and β-CDE conjugate (DS 1.3). Meanwhile, GUG-β-CDE conjugate (DS 1.8)/pDNA complex was likely to have high endosomal escaping ability and nuclear localization ability in A549 and RAW264.7 cells. In addition, the pDNA condensation and decondensation abilities of GUG-β-CDE conjugate (DS 1.8) were lower and higher than that of α-CDE conjugate (DS 1.2) or β-CDE conjugate (DS 1.3), respectively. These results suggest that high gene transfer activity of GUG-β-CDE conjugate (DS 1.8) could be, at least in part, attributed to high endosomal escaping ability, nuclear localization ability and suitable pDNA release from its complex. [Copyright &y& Elsevier]

In this study, we evaluated the polyamidoamine starburst dendrimer (dendrimer, generation 2: G2) conjugate with 6- O-α-(4- O-α-D-glucuronyl)-D-glucosyl-β-cyclodextrin (GUG-β-CDE (G2)) as a gene transfer carrier. The in vitro gene transfer activity of GUG-β-CDE (G2, degree of substitution (DS) of cyclodextrin (CyD) 1.8) was remarkably higher than that of dendrimer (G2) conjugate with α-CyD (α-CDE (G2, DS 1.2)) and that with β-CyD(β-CDE (G2, DS 1.3)) in A549 and RAW264.7 cells. The particle size, ζ-potential, DNase I-catalyzed degradation, and cellular association of plasmid DNA (pDNA) complex with GUG-β-CDE (G2, DS 1.8) were almost the same as those of the other CDEs. Fluorescent-labeled GUG-β-CDE (G2, DS 1.8) localized in the nucleus 6 h after transfection of its pDNA complex in A549 cells, suggesting that nuclear localization of pDNA complex with GUG-β-CDE (G2, DS 1.8), at least in part, contributes to its high gene transfer activity. GUG-β-CDE (G2, DS 1.8) provided higher gene transfer activity than α-CDE (G2, DS 1.2) and β-CDE (G2, DS 1.3) in kidney with negligible changes in blood chemistry values 12 h after intravenous injection of pDNA complexes with GUG-β-CDE (G2, DS 1.8) in mice. In conclusion, the present findings suggest that GUG-β-CDE (G2, DS 1.8) has the potential for a novel polymeric pDNA carrier in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Abstract: Arundic acid, (R)-(−)-2-propyloctanonic acid, is a novel neurological agent for intractable neurodegenerative diseases. However, arundic acid, an oily drug, has low aqueous solubility and severe bitter/irritating tastes. Consequently, these physicochemical properties of arundic acid need to be improved to develop its pharmaceutical preparations. In the present study, we evaluated whether parent cyclodextrins (CyDs) and 2-hydroxypropylated CyDs (HP-CyDs) can interact with arundic acid, and have powderization, solubilization and taste-masking properties. Of various CyDs, HP-β-CyD had the most potent solubilizing effect for arundic acid. UV and 1H NMR spectroscopic studies demonstrated that arundic acid formed inclusion complexes with CyDs at a molar ratio of 1:1 in solution. The complexation with CyDs changed an oily form of arundic acid to a solid form. The gustatory sensation studies indicate that of various CyDs, HP-β-CyD and γ-CyD showed the most significant taste-masking effects in solution and powders, respectively. HP-β-CyD significantly reduced the response of the electric potential caused by the adsorption of arundic acid to the taste sensor. These results suggest that hydrophilic CyDs have potential as multifunctional excipients for preparing solutions and powders containing arundic acid. [Copyright &y& Elsevier]

Abstract: 5-Fluorouracil-1-acetic acid (5-FUA) was prepared and covalently conjugated to β-cyclodextrin (β-CyD) through ester or amide linkage, and the drug release behavior of the conjugates in enzymatic solutions and rat cecal contents were investigated. The 5-FUA/β-CyD ester conjugate was slowly hydrolyzed to 5-FUA in aqueous solutions (half lives (t 1/2)=38 and 17h at pH 6.8 and 7.4, respectively, at 37°C), whereas the amide conjugate was hardly hydrolyzed at these physiological conditions, but hydrolyzed only in strong alkaline solutions (>0.1M NaOH) at 60°C. Both ester and amide conjugates were degraded in solutions of a sugar-degrading enzyme, α-amylase, to 5-FUA/maltose and triose conjugates, but the release of 5-FUA was only slight in α-amylase solutions. In solutions of an ester-hydrolyzing enzyme, carboxylic esterase, the ester conjugate was hydrolyzed to 5-FUA at the same rate as that in the absence of the enzyme, whereas the amide conjugate was not hydrolyzed by the enzyme. On the other hand, 5-FUA was rapidly released when the ester conjugate was firstly hydrolyzed by α-amylase, followed secondly by carboxylic esterase. The results indicated that the ester conjugate was hydrolyzed to 5-FUA in a consecutive manner, i.e. it was firstly hydrolyzed to the small saccharide conjugates, such as the maltose conjugate, by α-amylase, and the resulting small saccharide conjugates having less steric hindrance was susceptible to the action of carboxylic esterase, giving 5-FUA. The in vitro release behavior of the ester conjugate was clearly reflected in the hydrolysis in rat cecal contents and in the in vivo release after oral administration to rats. [Copyright &y& Elsevier]

Background and Purpose: Niemann-Pick disease type C (NPC) is a lysosomal storage disorder with disrupted intracellular cholesterol trafficking. A cyclic heptasaccharide, 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), is a cholesterol solubilizer that is being developed to treat NPC, but its ototoxicity and pulmonary toxicity remain important issues. We have characterized 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD), a cyclic octasaccharide with a larger cavity than HP-β-CD, as a candidate drug to treat NPC. However, the molecular target of HP-γ-CD with respect to NPC and its potential for clinical application are still unclear.Experimental Approach: We investigated the mode of interaction between HP-γ-CD and cholesterol by phase-solubility analysis, proton NMR spectroscopy and molecular dynamics simulations. We then evaluated the therapeutic effects of HP-γ-CD compared with HP-β-CD using cellular and murine NPC models. Mouse auditory and pulmonary function tests were also conducted.Key Results: HP-γ-CD solely formed a 1:1 inclusion complex with cholesterol with an affinity similar to that of HP-β-CD. In vitro, HP-γ-CD and HP-β-CD amelioration of NPC-related manifestations was almost equivalent at lower concentrations. However, at higher concentrations, the cholesterol inclusion mode of HP-β-CD shifted to the highly soluble 2:1 complex whereas that of HP-γ-CD maintained solely the 1:1 complex. The constant lower cholesterol solubilizing ability of HP-γ-CD conferred it with significantly reduced toxicity compared with HP-β-CD, but equal efficacy in treating a mouse model of NPC.Conclusions and Implications: HP-γ-CD can serve as a fine-tuned cholesterol solubilizer for the treatment of NPC with a wider safety margin than HP-β-CD in terms of ototoxicity and pulmonary toxicity. [ABSTRACT FROM AUTHOR]

Non-small cell lung cancer (NSCLC) is the leading cause of lung cancer-related deaths worldwide. Tumor-associated macrophages (TAMs), which can be polarized into tumor-promoting M2 phenotype, overexpress folate receptor beta (FRβ) and are associated with poor prognosis in NSCLC. In addition, calpain-2 (CAPN2) is overexpressed in NSCLC and is involved in tumor growth. To improve the anticancer efficacy of drugs and reduce their side effects in the treatment of NSCLC, it is important to develop smart drug delivery systems with specific targeting ability and controlled release mechanisms. In this study, FRβ-targeted pH-sensitive liposomes were designed as carriers to ensure efficient drug delivery and acid-responsive release in NSCLC cells. Folate-mediated targeting of FRβ in M2 TAMs and NSCLC cells effectively inhibited tumor growth and the stimulus-responsive drug release reduced the toxic side effects of the drug. The combination of doxycycline (anti-CAPN2) and docetaxel (anticancer drug) showed a synergistic inhibitory effect on tumor growth by suppressing CAPN2 expression. Unlabelled Image • Folate receptor β-targeting efficiently delivers drugs to non-small cell lung cancer. • Targeting of M2 tumor-associated macrophages effectively inhibits tumor growth. • Calpain-2 suppression shows synergistic inhibitory effects on tumor growth. • pH-responsive drug release reduces the toxic side effects of the drug. [ABSTRACT FROM AUTHOR]

Direct cytosolic delivery of the Cas9 ribonucleoprotein is the most promising method for inducing CRISPR-Cas9 genome editing in mammalian cells. Recently, we focused the movable properties of cyclodextrin-based polyrotaxanes (PRXs), which consist of numerous cyclodextrins threaded onto the axile molecule with bulky endcaps at both ends of the axile molecule, and developed aminated PRXs as multistep transformable carriers for Cas9 ribonucleoprotein, ensuring efficient complexation, cellular internalization, endosomal escape, release, and nuclear localization. This study reports the structural fine-tuning and structure-property relationship of multistep transformable PRXs for more efficient Cas9 ribonucleoprotein delivery. Among various PRXs, PRX derivatives with a longer molecular length (35 kDa polyethylene glycol as the axile molecule) and a low total degree of substitution (1.5 amino groups/α-cyclodextrins), as well as the modified ratio of two modified amines (cystamine and diethylenetriamine) = ≈1:1, exhibited the highest genome-editing efficacy and intracellular dynamics control. These structural properties are important for efficient endosomal escape and Cas9 RNP release. Furthermore, ligand-modified-β-CD, which can endow the ligand through complexation with PRX termini, improved the cellular uptake and genome-editing effects of the optimized PRX/Cas9 RNP in target cells. Thus, structural fine-tuning and the addition of ligand-modified-β-cyclodextrin enabled efficient genome editing by the Cas9 RNP. [Display omitted] [ABSTRACT FROM AUTHOR]

The blood-brain barrier (BBB) prevents the permeability of drugs into the brain, and as such limits the management of various brain diseases. To overcome this barrier, drug-encapsulating nanoparticles or vesicles, drug conjugates, and other types of drug delivery systems (DDSs) have been developed. However, the brain-targeting ability of nanoparticles or vesicles is still insufficient. Recently, among the various brain-targeting ligands previously studied for facilitating transcellular BBB transport, several sugar-appended nanocarriers for brain delivery were identified. Meanwhile, cyclodextrins (CyDs) have been used as nanocarriers for drug delivery since they can encapsulate hydrophobic compounds with high biocompatibility. Therefore, in this study, we created various sugar-appended β-cyclodextrins (β-CyDs) to discover novel brain-targeting ligands. As a result, of the six sugar-appended CyDs, lactose-appended β-CyD (Lac-β-CyD) showed greater cellular uptake in hCMEC/D3 cells, human brain microvascular endothelial cells, than other sugar-appended β-CyDs did. In addition, the permeability of Lac-β-CyD within the in vitro human BBB model was greater than that of other sugar-appended β-CyDs. Moreover, Lac-β-CyD significantly accumulated in the mouse brain after intravenous administration. Thus, Lac-β-CyD efficiently facilitated the accumulation of the model drug into the mouse brain. These findings suggest that Lac-β-CyD has the potential to be a novel carrier for drugs across the BBB. Unlabelled Image • Lactose-appended β-cyclodextrin (Lac-β-CyD), a novel drug carrier for brain, showed potent cellular uptake in human brain microvascular endothelial cells. In addition, the permeability of Lac-β-CyD within the in vitro human BBB model was greater than that of other sugar-appended β-CyDs. Moreover, Lac-β-CyD significantly accumulated in the mouse brain after intravenous administration. Thus, Lac-β-CyD efficiently facilitated the accumulation of the model drug into the mouse brain. These findings suggest that Lac-β-CyD has the potential to be a novel carrier for drugs across the BBB. [ABSTRACT FROM AUTHOR]

Transthyretin (TTR)-related familial amyloidotic polyneuropathy, which is induced by amyloidogenic transthyretin (ATTR), is characterized by systemic accumulation of amyloid fibrils. Although it is believed that protein misfolding of monomeric form of TTR is a rate-limiting step for TTR amyloid formation, no effective therapy targeting this misfolding step is available. Our recent studies revealed that cyclodextrins (CyDs), cyclic oligosaccharides composed of glucose units, might interact with TTR and prevent the protein misfolding. In this study, we focused on and elucidated the inhibitory effect of 6-O- a-(4-O-α-d-Glucuronyl)-d-glucosyl-β-CyD (GUG-β-CyD) on TTR amyloid formation. Tryptophan (Trp) fluorescence and 1H-NMR spectroscopy analyses indicated that GUG-β-CyD stabilized TTR conformation via interaction with the hydrophobic amino acids of TTR. Moreover, GUG-β-CyD suppressed TTR deposition in transgenic rats possessing a human ATTR V30M gene in vivo. Collectively, these data indicate that GUG-β-CyD may inhibit TTR misfolding by stabilizing its conformation, which, in turn, suppresses TTR amyloid formation. [ABSTRACT FROM AUTHOR]