Single-step preparation of topological gels using vinyl-modified β-cyclodextrin as a figure-of-six cross-linker

Herein, we have proposed a single-step preparation of topological gels using vinyl-modified β-cyclodextrin (V-β-CyD) and isoprene. Copolymerization of V-β-CyD and isoprene in an aqueous solution resulted in gelation due to V-β-CyD acting as a novel type of copolymer chain cross-linker. The vinyl moiety of V-β-CyD becomes a part of the copolymer, while the β-CyD moiety of V-β-CyD simultaneously incorporates the isoprene component of the copolymer. V-β-CyD is capable of two different modes of cross-linking at each end, i.e., chemically bonding and mechanically interlocking. Due to the shape of the cross-linking point, we refer to it as figure-of-six cross-linking. Nuclear magnetic resonance analysis showed that the gel contained V-β-CyD and isoprene in an approximately 1:0.3 stoichiometry. The relatively high content of β-CyD was reflected in the character of the gel; the gel swelled in dimethylformamide which is a good solvent of β-CyD. A fluorometric analysis using 6-(p-toluidino)-2-naphthalenesulfonic acid showed that the appended β-CyD was able to accommodate guest molecules. Introduction of an additional vinyl monomer into the gel was also successful. Addition of 4-vinylphenylboronic acid to the preparation procedure yielded a sugar-responsive gel that swelled in the presence of d-fructose.     

Release control of fragrances by complexation with β-cyclodextrin and its derivatives

The release control of fragrances, benzyl acetate (BA), citral (CR), linalool (LL), citronellol (CL) and linalyl acetate (LA), was conducted using β-cyclodextrin (β-CyD), 2-hydroxypropyl-β-CyD (HP-β-CyD) and 2,6-di-O-methyl β-CyD (DM-β-CyD). The release rate of the fragrances from 30% ethanol/water solution was significantly suppressed by the complexation with these CyDs, and the suppressing effect increased in the order of β-CyD?<?HP-β-CyD?<?DM-β-CyD. The concentration-dependent change of the release rate was quantitatively analyzed to obtain the stability constant (Kc) of the fragrance-CyD complexes. These Kc values were in good agreement with those determined by the solubility method. The results suggest that the release of fragrances can be prolonged by the complexation with β-CyDs and their effects can be controlled by choosing appropriate CyD derivatives with higher Kc values and by setting proper concentrations of the host molecules.     

Inhibitory Effects of 2,6-Di-O-methyl-3-O-acetyl-β-cyclodextrins with Various Degrees of Substitution of Acetyl Group on Macrophage Activation and Endotoxin Shock Induced by Lipopolysaccharide

The effects of 2,6-di-O-methyl-3-O-acetyl-β-cyclodextrins (DMA-β-CyD) with various degrees of substitution (DS) of an acetyl group of 1.5, 3.8, 6.3 and 7, which are abbreviated to DMA2-β-CyD, DMA4-β-CyD, DMA6-β-CyD and DMA7-β-CyD, respectively, on murine macrophage activation and endotoxin shock induced by lipopolysaccharide (LPS) were examined. Of four DMA-β-CyDs used in the present study, cytotoxicity of DMA-β-CyDs in RAW264.7 cells, a murine macrophage-like cell line, decreased with an increase in the DS values of DMA-β-CyD, and DMA7-β-CyD had no cytotoxicity on RAW264.7 cells up to 100 mM. DMA2-β-CyD and DMA7-β-CyD at the concentration of 5 mM had greater inhibitory effects on nitric oxide (NO) production in RAW264.7 cells stimulated with LPS than DMA4-β-CyD and DMA6-β-CyD. In addition, these inhibitory effects of DMA2-β-CyD and DMA7-β-CyD were concentration-dependent. In the in vivo study, all of the mice died within 12 h after intraperitoneal administration of the solution containing LPS and d-galactosamine. When 100 mM DMA7-β-CyD was concomitantly administered with both LPS and d-galactosamine intraperitoneally in mice, the survival rate significantly increased, but DMA4-β-CyD and DMA6-β-CyD did not. In conclusion, we revealed that DS values of DMA-β-CyDs strikingly affect not only the cytotoxic activity but also the inhibitory effects of LPS-induced NO production in RAW264.7 cells and fatality of endotoxin shock mice induced by LPS and d-galactosamine. These results suggest the potential use of DMA7-β-CyD as an antagonist of LPS-induced endotoxin shock.     

Complexation studies of pioglitazone hydrochloride and β-cyclodextrin: NMR (1H, ROESY) spectroscopic study in solution

Pioglitazone hydrochloride (PIO) is an agonist of the peroxisome proliferator-activated receptor γ (PPARγ), used to treat diabetes. ¹H-NMR spectroscopic analysis of varying ratios of β-cyclodextrin (β-CyD) and PIO in D₂O confirmed the formation of β-CyD–PIO inclusion complex in aqueous solution. The 1:1 stoichiometry of β-CyD–PIO inclusion complex was determined by Scott’s plot method and binding constant (K _a ) was calculated to be 155 M^?1. 2D ROESY experiments confirmed that the phenyl ring of PIO act as a guest and deeply penetrate in β-CyD cavity from wider as well as narrower rim side and form two 1:1 stable inclusion complexes. Some of the PIO protons exhibited splitting, in the presence of β-CyD, indicating chiral differentiation of PIO by β-CyD.     

Physicochemical characterization of coumestrol/β-cyclodextrins inclusion complexes by UV–vis and FTIR-ATR spectroscopies

The inlcusion behaviour of unsubstituted β-cyclodextrin (β-CyD) and (2-hydroxypropyl)-β-cyclodextrin (HP-β-CyD), in solution and solid state was studied with regards to a poorly water-soluble bioflavonoid, coumestrol (Coum), namely 7,12-dihydroxycoumestan, well-known for its anti-oxidant, anti-inflammatory, anti-fungal and anti-viral activities. Phase-solubility measurements were performed to evaluate in solution the complexation of the two cyclodextrins, i.e. β-CyD and HP-β-CyD. The stoichiometry and stability constants of the Coum/β-CyD and Coum/HP-β-CyD complexes were calculated by the phase-solubility method, after which drug–cyclodextrin solid systems were prepared by co-precipitation. In solid phase, the formation of inclusion complexes was confirmed by Fourier transform infrared spectroscopy in attenuated total reflectance (FTIR-ATR) geometry. In particular, complexation mechanisms were explained by the significant differences revealed in the FTIR-ATR spectra of physical mixtures with respect to those of the complexes; the use of deconvolution and curve fitting was determinant.     

Combined use of cyclodextrins and hydroxypropylmethylcellulose stearoxy ether (Sangelose®) for the preparation of orally disintegrating tablets of type-2 antidiabetes agent glimepiride

Despite recent advances in the formulation of orally disintegrating tablets (ODTs), the efforts to enhance the swallowing of the drug after disintegration have been limited. In this study, the feasibility of the combined use of cyclodextrins (CyDs) and a functional drug carrier, hydroxypropylmethylcellulose stearoxy ether (Sangelose^®) was investigated to improve usability of ODTs. Glimepiride, a potent third generation hypoglycemic agent for type 2 diabetes was used as a model drug, because it is poorly water-soluble and elimination half life is fairly short. The direct compression method was employed for the preparation of glimepiride tablets, containing CyDs and Sangelose^®, and various characteristics of the tablets were examined. In the cases of α-CyD and β-CyD, a short disintegration time with an appropriate hardness was obtained, complying with ODT criteria. On the other hand, γ-CyD, HP-β-CyD and HB-β-CyD increased in the hardness and disintegration time of the tablets. The rheological evaluation revealed that CyDs, except γ-CyD, significantly reduced the viscosity of the fluids after disintegration of the tablets, suggesting an ease of swallowing. This was ascribable to the complexation of the hydrophobic stearoyl moiety of Sangelose^® with CyDs after dissolution, leading to the inhibition of the polymer–polymer interaction of Sangelose^® and to the decrease in viscosity of the solution. The interaction of glimepiride with α- and β-CyDs was studied by the solubility method, demonstrating that glimepiride formed water-soluble complexes with these CyDs. Results obtained here suggested that α-CyD and β-CyD can be particularly useful for the Sangelose^®-based ODT formulation, compared to γ-CyD, HP-β-CyD and HB-β-CyD, because of the short disintegration time of the tablets containing α-CyD and β-CyD, their shear-thinning effect on Sangelose^® solutions and their solubility enhancing effect on the drug.     

Phase solubility analysis in studying the interaction of nifedipine with selected cyclodextrins in aqueous solution

The solubilizing potential and complexing tendencies of six cyclodextrins (CyD) with nifedipine in aqueous solution were evaluated using phase solubility methods. Solubility curves of nifedipine with -CyD, 2-hydroxypropyl--CyD (2HP--CyD) and 2-hydroxypropyl--cyclodextrin (2HP--CyD) were classified as type A_L, while for heptakis (2,6-dimethyl)--CyD (DIMEB), randomly methylated--CyD (RAMEB) and -CyD, A_p type phase behaviour was observed. Stability constants, calculated from phase solubility diagrams, decreased in the order: DIMEB > RAMEB > -CyD > 21HP--CyD > -CyD > 2HP--CyD.     

MC8 Peptide-Mediated Her-2 Receptor Targeting Based on PEI-β-CyD as Gene Delivery Vector

A novel vector with high gene delivery efficiency and special cell targeting ability was developed using a good strategy that utilized low molecular weight polyethylenimine (PEI; molecular weight, 600 KDa [PEI600]) cross-linked to β-cyclodextrin (β-CyD) via a facile synthetic route. Human epidermal growth factor receptor 2 (Her-2) are highly expressed in a variety of human cancer cells and are potential targets for cancer therapy. MC8 peptides, which have been proven to combine especially with Her-2 on cell membranes were coupled to PEI-β-CyD using N-succinimidyl-3-(2-pyridyldithio) propionate as a linker. The ratios of PEI600, β-CyD, and peptide were calculated based on proton integral values obtained from the ¹H-NMR spectra of the resulting products. Electron microscope observations showed that MC8-PEI-β-CyD can efficiently condense plasmid DNA (pDNA) into nanoparticles of about 200 nm, and MTT assays suggested the decreased toxicity of the polymer. Experiments on gene delivery efficiency in vitro showed that MC8-PEI-β-CyD/pDNA polyplexes had significantly greater transgene activities than PEI-β-CyD/pDNA in the Skov3 and A549 cells, which positively expressed Her-2, whereas, no such effect was observed in the MCF-7 cells, which negatively expressed Her-2. Our current research indicated that the synthesized nonviral vector shows improved gene delivery efficiency and targeting specificity in Her-2 positive cells.     

Improvement of pharmaceutical potential of all-trans retinoic acid with hydroxypropyl-��-cyclodextrin

2-Hydroxypropyl-??-cyclodextrin (HP-??-CyD) includes all-trans retinoic acid (RA), covering the double-bond area of RA with substituted hydroxypropyl groups on CyD ring, as proved by the nuclear Overhauser effect (NOE) between methylene protons on the hydroxypropyl groups and the proton on RA. The formation of an inclusion complex results in hydrophilicity and stability. The effect of RA/HP-??-CyD and that of RA without HP-??-CyD on wrinkle scores and skin elasticity during skin treatment were identical, and the cutaneous stimulus was reduced comparing with RA. The results indicated that the RA/HP-??-CyD complex should help to realize new approaches in skin rejuvenation therapy.     

Fluorescence spectra of bithiophene and terthiophene single crystals and of their isolated molecules in cyclodextrin

In order to examine the effect of subsumption space of β- and γ-cyclodextrin (CyD) on the photophysics of oligothiophenes, 2, 2′-bithiophene (BT) and 2, 2′: 5′, 2″-terthiophene (TT), the fluorescence spectra were compared with those of the single crystals (SC) at 15, and room temperature (RT). Both the numbers of BT included in β- and γ-CyD are twin (BT₂). The numbers of TT included in β- and γ-CyD are unit (TT₁) and twin (TT₂), respectively. Electronic excitation of BT encapsulated in β- and γ-CyD gives similar fluorescence spectra, showing bathochromic shift compared with that of BT single crystal, (BT)_SC. The observation that the fluorescence spectra of encapsulated BT₂ are similar to the spectra of its THF solution suggests the configuration of BT₂ in β- and γ-CyD should be face-to-face configuration (BT₂)_∥. On the contrary, TT in β- and in γ-CyD afford quite different fluorescence spectra. Encapsulated TT in β-CyD exhibits the hypsochromic shift of fluorescence maxima compared to that of TT single crystal, (TT)_SC. While the bathochromically shifted fluorescence spectra of TT₂ in γ-CyD is also ascribed to the face-to-face configuration (TT₂)_∥ as in the case of BT₂ in γ-CyD. Fluorescence spectra show the excited ground state complex of BT₂ and TT₂ in γ-CyD.     

A 2 : 2 stilbeneboronic acid-γ-cyclodextrin fluorescent ensemble highly selective for glucose in aqueous solutions

By designing a long and hydrophobic stilbeneboronic acid (STDBA) and allowing it to be included within the cavity of γ-cyclodextrin (γ-CyD) we succeeded in developing a fluorescent 2 : 2 STDBA-γ-CyD ensemble that shows a selective and sensitive response toward glucose in aqueous solutions, with satisfactory application in artificial urine samples.     

Design and evaluation of folate-appended methyl-��-cyclodextrin as a new antitumor agent

Methyl-??-cyclodextrin (M-??-CyD) is widely used as a raft disrupting agent through extraction of cholesterol from lipid rafts which are highly expressed in cell membranes of tumor cells, but it does not have tumor cell-selective action. Meanwhile, the widespread use of folic acid (FA) as a tumor-targeting ligand has been known, because folate receptor (FR) overexpresses in various kinds of epithelial tumor cells. In the present study, in order to obtain more tumor cell-selectivity and antitumor activity of M-??-CyD, we designed folate-appended M-??-CyD (FA-M-??-CyD), and evaluated its physicochemical properties and antitumor activity. The ¹H-NMR study demonstrated that FA-M-??-CyD having average degree of substitution of FA (DSF) of 1.0 was prepared. In addition, FA-M-??-CyD (DSF 1.0) was found to be amorphous in a solid state and surface-active. Importantly, FA-M-??-CyD (DSF 1.0) had potent cytotoxicity, compared to M-??-CyD in KB cells, but not in A549 cells. These results suggest that FA-M-??-CyD (DSF 1.0) has the potential as a novel antitumor agent.     

Some Pharmaceutical Properties of a New Branched Cyclodextrin, 6-O-α-(4-O- α-D-Glucuronyl)-D-glucosylβ-cyclodextrin

Some physicochemical and biological properties of a new branched cyclodextrin, 6-O--(4-O--d-glucuronyl)-d-glucosyl--cyclodextrin GUG--CyD) were investigated. Further, theinteraction of GUG--CyD with several drugs was studied by the solubility and spectroscopic methods, and compared with those of parent -CyD and 6-O--maltosyl--CyD(₂--CyD).The hemolytic activity of GUG--CyD on rabbit erythrocytes was lower than those of -CyD and ₂--CyD. GUG--CyD and ₂--CyD showed negligible cytotoxicity on Caco-2 cells up to at least 0.1 M. The inclusion ability of GUG--CyD to neutral and acidic drugs was comparable to or slightly smaller than those of -CyD and ₂--CyD, probably because of a steric hindrance of the branched sugar. On the other hand, GUG--CyD showed greater affinity for the basic drugs, compared with -CyD and ₂--CyD, owing to the electrostatic interaction of its carboxylate anion with positive charge of basic drugs. Thus GUG--CyD may be useful as a safe solubilizing agent particularly for basic drugs.     

Interaction of Natural and Modified β-Cyclodextrins with a Biological Membrane Model of Dipalmitoylphosphatidylcholine

Lipid vesicles made up of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were used as a biological membrane model to investigate the interaction between natural and modified β-cyclodextrins and these membrane bilayers. Differential scanning calorimetry was used to study the thermotropic behavior of the DPPC vesicles and any change caused by the presence of cyclodextrins. The presence of dimethyl-β-cyclodextrin (DM-β-CyD) triggered a reduction in the enthalpy values related to the main transition peak from gel state to liquid crystal phase of DPPC aqueous dispersions, as a function of the DM-β-CyD molar fraction: the larger the amount of DM-β-CyD, the greater the reduction in ΔHvalues. This effect was probably due to the ability of DM-β-CyD to extract and to complex the DPPC molecules forming the phospholipid vesicles. The presence of β-cyclodextrin (β-CyD) or hydroxypropyl-β-cyclodextrin (HP-β-CyD) caused no particular alteration in the thermotropic parameters of DPPC vesicles, whereas trimethyl-β-cyclodextrin (TM-β-CyD) at molar fractions higher than 0.12 caused broadening of the transition peak due to a possible interaction with the hydrophobic part of the bilayers. Experiments on DPPC–cholesterol (10 mol%) vesicles showed the capability of β-CyD and TM-β-CyD to extract cholesterol from the ordered bilayer structures, triggering an alteration in the lipid constituents of the membranes. HP-β-CyD caused no variation in the thermotropic parameters of the DPPC–cholesterol (10 mol%) vesicles. The findings show that HP-β-CyD seems the most suitable molecular drug carrier forin vivoadministration.     

Potential use of polypseudorotaxanes of pegylated polyamidoamine dendrimer with cyclodextrins as novel sustained release systems for DNA

In this study, we demonstrated the potential use of polypseudorotaxanes (PPRXs) of polyethylene glycol (PEG, molecular weight: 2000)-grafted polyamidoamine dendrimer (PEG-dendrimer) with cyclodextrins (CyDs) as novel sustained release systems for plasmid DNA (pDNA). PEG-dendrimer/pDNA complex formed PPRXs with α-CyD and γ-CyD solutions, but not with β-CyD solution. In the PEG-dendrimer/CyDs PPRXs systems, 17.9 mol of α-CyD and 8.8 mol of γ-CyD were involved in the PPRXs formation with one PEG chain by α-CyD and γ-CyD, respectively. In addition, the CyDs PPRX formation provided the sustained release of pDNA from PEG-dendrimer complex with pDNA at least 72 h in vitro. In addition, the release of pDNA from CyDs PPRX retarded as the dissolution medium volume decreased. These results suggest that the PEG-dendrimer/CyD PPRX systems can work as a sustained DNA release system, and the PPRX formation with CyDs may be useful as a sustained drug delivery technique for other pegylated polymers.     

Prominent Solubilizing Effect of 2-Hydroxypropyl-β-cyclodextrin on a New Thiazolidine Derivative (FPFS-410) with Antidiabetic and Lipid-lowering Activities through Inclusion Complex Formation

2-(N-Cyanoimino)-5-{(E)-4-styrylbenzylidene}-4-oxothiazolidine (FPFS-410) is a newly synthesized thiazolidine derivative having not only antidiabetic but also lipid-lowering activities. However, this compound has an extremely low aqueous solubility (2.8×10⁻⁸ M in phosphate buffer at 25 °C). In this study, we attempted to improve the solubility of FPFS-410 in water, by means of the complexation with 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD). Further, the interaction of FPFS-410 with HP-β-CyD in 50% v/v methanol/water mixed solution was investigated by ultraviolet and ¹H-nuclear magnetic resonance (NMR) spectroscopic methods, because the solubility of FPFS-410 in water was too low to study quantitatively the interaction. The results of the solubility method indicated that HP-β-CyD had a markedly high solubilizing ability to FPFS-410, e.g., the solubility of the compound was increased 200,000-fold by the addition of 40 mM HP-β-CyD. The continuous variation plot of the FPFS-410/HP-β-CyD system in 50% v/v methanol/water solution gave a maximum at a host/guest molar ratio of 1:1. ¹H-NMR spectroscopic studies suggested that the stilbene moiety of FPFS-410 is preferably included in the HP-β-CyD cavity to form the 1:1 complex in 50% v/v methanol/water solution. The present results suggest that HP-β-CyD is useful for improvement of the oral bioavailability of FPFS-410.     

Molecular imprinting of cyclodextrin to physiologically active oligopeptides in water

β-Cyclodextrin (β-CyD)-based polymeric receptors for γ-endorphin (γ-endor, an opioid heptadecapeptide) were prepared using the molecular imprinting method. When mono-3-(N-acrylamido)-3-deoxy-β-CyD bearing a vinyl group in the secondary hydroxyl side of the cavity of β-CyD was polymerised in water in the presence of γ-endor, the binding activity of the β-CyD polymer to this peptide in water was enormously promoted by the imprinting. By contrast, the bindings towards methionine–enkephalin (N-terminal pentapeptide of γ-endor) and its homologue leucine–enkephalin were suppressed. Thus, the binding of γ-endor by the imprinted polymer was highly selective. The imprinting towards γ-endor was also successful with the use of the β-CyD monomer bearing a vinyl group in the primary hydroxyl side of the cavity, although the recognition was less strict. Various factors affecting the imprinting efficiency (kinds of β-CyD vinyl monomer and template, as well as the pH of imprinting mixture) are discussed.     

Improvement of pharmaceutical properties of insulin through conjugation with glucuronylglucosyl-β-cyclodextrin

Recently, a large number of peptides and proteins have been utilized as active pharmaceutical ingredients in the clinical field. However, the stability of peptide and protein drugs is often low. In addition, some peptides and proteins adsorb onto glass or polypropylene tube. In the present study, to improve these pharmaceutical properties of peptides and proteins, we newly prepared glucuronylglucosyl-β-cyclodextrin (GUG-β-CyD) conjugate with insulin, a model protein drug, and evaluated its enzymatic or thermal stability and adsorption onto glass or polypropylene tube. The insulin conjugate with GUG-β-CyD was successfully prepared by condensation of amine group of insulin and carboxyl group of GUG-β-CyD. Circular dichroism spectra showed that the secondary structure of insulin in this conjugate was retained. Adsorption of insulin onto glass or polypropylene tube was decreased by the conjugation with GUG-β-CyD. Moreover, enzymatic and thermal stabilities of the conjugate were higher than those of insulin and the mixture of insulin and GUG-β-CyD. These results suggest that insulin conjugation with GUG-β-CyD could improve the pharmaceutical properties of insulin.     

Glucose recognition by a supramolecular complex of boronic acid fluorophore with boronic acid-modified cyclodextrin in water

A boronic acid fluorophore (C1-APB)/boronic acid-modified γ-cyclodextrin (3-PB-γ-CyD) complex as a supramolecular sensor has been designed for selective glucose recognition in water. The fluorescent response behavior of the C1-APB/3-PB-γ-CyD complex under various pH conditions revealed that a C1-APB/3-PB-γ-CyD complex solution containing glucose showed a large increase in the fluorescence intensity under alkaline pH conditions. In contrast, only small increases in the fluorescence intensity were noted for fructose and without sugar solutions. The observed response selectivity for the C1-APB/3-PB-γ-CyD complex was on the order of glucose > galactose, mannose > fructose. The evidence on a large value of the inclusion constant (K(L·CyD) = 6.5 × 10(3) M(-1)), a marked broadening of the (1)H NMR spectra, and an enhancement of induced circular dichloism (ICD) intensity for the C1-APB/3-PB-γ-CyD complex by glucose binding supported the multi-point interaction of the C1-APB/3-PB-γ-CyD complex with glucose. These results demonstrated that the C1-APB/3-PB-γ-CyD complex functioned as an efficient supramolecular sensor for selective glucose recognition in water.     

Involvement of mitophagy-mediated cell death in colon cancer cells by folate-appended methyl-β-cyclodextrin

In recent years, colorectal cancer has gained great attention among various types of cancers. We previously synthesized folate-appended methyl-β-cyclodextrin (FA-M-β-CyD) as a novel autophagic antitumor agent. In this study, to further elucidate the impact of FA-M-β-CyD as an antitumor agent, we evaluated cytotoxic activity and the antiproliferative effect in colon cancer cells and colorectal cancer model mice, respectively. As a result, FA-M-β-CyD showed potent cytotoxic activity in HCT116 cells, a human colon cancer cell line, through folate receptor-α (FR-α)-mediated cellular uptake. In addition, FA-M-β-CyD elicited autophagosome formation and induced mitophagy in HCT116 cells. Importantly, FA-M-β-CyD drastically reduced the number of tumor polyps in colorectal cancer model mice induced by azoxymethane/dextran sodium sulfate after intravenous injections once a week for 4 weeks. These results suggest that FA-M-β-CyD has the antiproliferative effect in the colorectal cancer, due to the FR-α-mediated endocytosis and mitophagy induction.