Re-exposure of chitosan by an inhalable microsphere providing the re-education of TAMs for lung cancer treatment with assistant from sustained H2S generation.

The re-education of tumor-associated macrophages (TAMs) is an effective strategy to inhibit the growth and metastasis of lung cancer. We have reported that chitosan could re-educate the TAMs and then inhibit cancer metastasis; however, the re-exposure of chitosan from the chemical corona on their surface is critical for this effect. In this study, a strategy was proposed to re-expose the chitosan from chemical corona, and a sustained H2S generation was applied to enhance the immunotherapy of chitosan. To achieve this objective, an inhalable microsphere (namely F/Fm) was designed, which could be degraded by the matrix metalloproteinase in lung cancer, releasing two kinds of nanoparticles; in an external magnetic field, these nanoparticles can aggregate with each other, and ß-cyclodextrin on the surface of one nanoparticle can be hydrolyzed by amylase on the surface of another nanoparticle, leading to the re-exposure of chitosan in the inner layer of ß-cyclodextrin and the release of diallyl trisulfide for H2S generation. In vitro, the expression of CD86 and secretion of TNF-α by TAMs was increased by F/Fm, proving the re-education of TAMs, and the apoptosis of A549 cells was promoted with the migration and invasion being inhibited. In the Lewis lung carcinoma-bearing mouse, the F/Fm re-educated the TAMs and provided a sustained generation of H2S in the region of lung cancer, effectively inhibiting the growth and metastasis of lung cancer cells. This work provides a new strategy for the treatment of lung cancer in combination of re-education of TAMs by chitosan and the adjuvant chemotherapy by H2S.

Investigation of 2-Hydroxypropyl-ß-Cyclodextrin Treatment in a Neuronal-Like Cell Model of Niemann-Pick Type C Using Quantitative Proteomics.

Niemann-Pick, type C (NPC) is a fatal, neurovisceral lysosomal storage disorder with progressive neurodegeneration and no FDA-approved therapy. Significant efforts have been focused on the development of therapeutic options, and 2-hydroxypropyl-ß-cyclodextrin (HP-b-CD) has emerged as a promising candidate. In cell culture, HP-b-CD ameliorates cholesterol storage in endo/lysosomes, a hallmark of the disorder. Furthermore, in animal studies, treatment with HP-b-CD delays neurodegeneration and extends lifespan. While HP-b-CD has been promising in vitro and in vivo, a clear understanding of the mechanism(s) of action is lacking. Utilizing a neuron-like cell culture model of SH-SY5Y differentiated cells and U18666A to induce the NPC phenotype, we report here a large-scale mass-spectrometry-based proteomic study to evaluate proteome changes upon treatment with these small molecules. In this study, we show that differentiated SH-SY5Y cells display morphological changes representative of neuronal-like cells along with increased levels of proliferation markers. Inhibition of the NPC cholesterol transporter 1 protein by U18666A resulted in increased levels of known NPC markers including SCARB2/LIMP2 and LAMP2. Finally, investigation of HP-b-CD treatment was performed where we observe that, although HP-b-CD reduces cholesterol storage, levels of NPC1 and NPC2 are not normalized to control levels. This finding further supports the need for a proteostasis strategy for NPC drug development. Moreover, proteins that were dysregulated in the U18666A model of NPC and normalized to control levels suggest that HP-b-CD promotes exocytosis in this neuron-like model. Utilizing state of the art mass spectrometry analysis, these data demonstrate newly reported changes with pharmacological perturbations related to NPC disease and provide insight into the mechanisms of HP-b-CD as a potential therapeutic.

Evidence of redox imbalance and mitochondrial dysfunction in Niemann-Pick type C 1 patients: the in vitro effect of combined therapy with antioxidants and ß-cyclodextrin nanoparticles.

Niemann-Pick C disease (NPC) is an autosomal recessive genetic disorder resulting from mutation in one of two cholesterol transport genes: NPC1 or NPC2, causing accumulation of unesterified cholesterol, together with glycosphingolipids, within the endosomal/lysosomal compartment of cells. The result is a severe disease in both multiple peripheral organs and the central nervous system, causing neurodegeneration and early death. However, the pathophysiological mechanisms of NPC1 remain poorly understood. Recent studies have shown that the primary lysosomal defect found in fibroblasts from NPC1 patients is accompanied by a deregulation of mitochondrial organization and function. There is currently no cure for NPC1, but recently the potential of ß-cyclodextrin (ß-CD) for the treatment of the disease was discovered, which resulted in the redistribution of cholesterol from subcellular compartments to the circulation and increased longevity in an animal model of NPC1. Considering the above, the present work evaluated the in vitro therapeutic potential of ß-CD to reduce cholesterol in fibroblasts from NPC1 patients. ß-CD was used in its free and nanoparticulate form. We also evaluated the ß-CD potential to restore mitochondrial functions, as well as the beneficial combined effects of treatment with antioxidants N-Acetylcysteine (NAC) and Coenzyme Q10 (CoQ10). Besides, we evaluated oxidative and nitrative stress parameters in NPC1 patients. We showed that oxidative and nitrative stress could contribute to the pathophysiology of NPC1, as the levels of lipoperoxidation and the nitrite and nitrate levels were increased in these patients when compared to healthy individuals, as well as DNA damage. The nanoparticles containing ß-CD reduced the cholesterol accumulated in the NPC1 fibroblasts. This result was potentiated by the concomitant use of the nanoparticles with the antioxidants NAC and CoQ10 compared to those presented by healthy individuals cells ́. In addition, treatments combining ß-CD nanoparticles and antioxidants could reduce mitochondrial oxidative stress, demonstrating advantages compared to free ß-CD. The results obtained are promising regarding the combined use of ß-CD loaded nanoparticles and antioxidants in the treatment of NPC1 disease.

Mitigating RANKL-induced cholesterol overload in macrophages with ß-cyclodextrin-threaded polyrotaxanes suppresses osteoclastogenesis.

Free cholesterol acts as an endogenous agonist for estrogen-related receptor α (ERRα), a nuclear receptor that regulates osteoclastogenesis. Because stimulation of macrophages with receptor activator of nuclear factor κB ligand (RANKL) induces an overload of free cholesterol and activates ERRα, we hypothesized that direct removal of cellular cholesterol would suppress osteoclastogenesis. In this study, the effect of 2-hydroxypropyl ß-cyclodextrin (HP-ß-CD), a highly water-soluble cyclic glucopyranose, and ß-CD-threaded polyrotaxanes (PRXs), supramolecular polymers designed to release threaded ß-CDs in acidic lysosomes, on RANKL-induced cholesterol overload and osteoclast differentiation of murine macrophage-like RAW264.7 cells were investigated. PRXs suppressed RANKL-induced cholesterol overload. Additionally, RANKL-induced osteoclast differentiation of RAW264.7 cells was inhibited by PRXs. In contrast, HP-ß-CD did not reduce cholesterol levels or inhibit osteoclast differentiation in RAW264.7 cells. Gene expression analysis of osteoclast markers suggested that PRXs suppress only the early stage of osteoclast differentiation, as PRXs cannot be internalized into multinucleated osteoclasts. However, modification of PRXs with cell-penetrating peptides facilitated their cellular uptake into multinucleated osteoclasts and inhibited osteoclast maturation. Thus, PRXs are promising candidates for inhibiting osteoclast differentiation by suppressing cholesterol overload and may be useful for treating osteoporosis or other bone defects caused by the overactivity of osteoclasts.

Medium optimization for submerged fermentative production of ß-cyclodextrin glucosyltransferase by isolated novel alkalihalophilic Bacillus sp. NCIM 5799 using statistical approach.

ß-cyclodextrin glucosyltransferase (ß-CGTase) is an essential enzyme to catalyse the biotransformation of starch into ß-cyclodextrins (ß-CD). ß-CD has widespread applications in the biomedical, pharmaceutical and food industries. The present study focused on ß-CGTase production using an efficient natural microbial strain and statistical production optimization for enhanced production. The isolated organism Bacillus sp. NCIM 5799 was found to be 5 µm short bacilli under FE-SEM and alkalihalophilic in nature. The ß-CGTase production was optimized using a combination of Plackett-Burman design (PBD) and Central Composite Design-Response Surface Methodology (CCD-RSM). On PBD screening Na2 CO3 , peptone and MgSO4 .7H2 O were found to be significant for optimal ß-CGTase production, whereas the soluble starch and K2 HPO4 concentrations were found to be nonsignificant for ß-CGTase production. The significant factors obtained after PBD were further optimized using CCD-RSM design. Peptone was found to have a significant interaction effect with Na2 CO3 , and MgSO4 ·7H2 O and Na2 CO3 exhibited a significant effect on the production of CGTase. The production of ß-CGTase was enhanced in the presence of peptone (3%) and Na2 CO3 (0·8%). CGTase production obtained was 156·76 U/ml when optimized using CCD-RSM. The final optimized medium (RSM) shows 7·7- and 5·4-fold high productions as compared to un-optimized and one factor at a time production media.

The Biological Fate of Pharmaceutical Excipient ß-Cyclodextrin: Pharmacokinetics, Tissue Distribution, Excretion, and Metabolism of ß-Cyclodextrin in Rats.

ß-cyclodextrin has a unique annular hollow ultrastructure that allows encapsulation of various poorly water-soluble drugs in the resulting cavity, thereby increasing drug stability. As a bioactive molecule, the metabolism of ß-cyclodextrin is mainly completed by the flora in the colon, which can interact with API. In this study, understanding the in vivo fate of ß-cyclodextrin, a LC-MS/MS method was developed to facilitate simultaneous quantitative analysis of pharmaceutical excipient ß-cyclodextrin and API dextromethorphan hydrobromide. The established method had been effectively used to study the pharmacokinetics, tissue distribution, excretion, and metabolism of ß-cyclodextrin after oral administration in rats. Results showed that ß-cyclodextrin was almost wholly removed from rat plasma within 36 h, and high concentrations of ß-cyclodextrin distributed hastily to organs with increased blood flow velocities such as the spleen, liver, and kidney after administration. The excretion of intact ß-cyclodextrin to urine and feces was lower than the administration dose. It can be speculated that ß-cyclodextrin metabolized to maltodextrin, which was further metabolized, absorbed, and eventually discharged in the form of CO2 and H2O. Results proved that ß-cyclodextrin, with relative low accumulation in the body, had good safety. The results will assist further study of the design and safety evaluation of adjuvant ß-cyclodextrin and promote its clinical development.

Insulin Complexation with Cyclodextrins-A Molecular Modeling Approach.

Around 5% of the population of the world is affected with the disease called diabetes mellitus. The main medication of the diabetes is the insulin; the active form is the insulin monomer, which is an instable molecule, because the long storage time, or the high temperature, can cause the monomer insulin to adapt an alternative fold, rich in ß-sheets, which is pharmaceutically inactive. The aim of this study is to form different insulin complexes with all the cyclodextrin used for pharmaceutical excipients (native cyclodextrin, methyl, hydroxyethyl, hydroxypropyl and sulfobutylether substituted ß-cyclodextrin), in silico condition, with the AutoDock molecular modeling program, to determine the best type of cyclodextrin or cyclodextrin derivate to form a complex with an insulin monomer, to predict the molar ratio, the conformation of the complex, and the intermolecular hydrogen bonds formed between the cyclodextrin and the insulin. From the results calculated by the AutoDock program it can be predicted that insulin can make a stable complex with 5-7 molecules of hydroxypropyl-ß-cyclodextrin or sulfobutylether-ß-cyclodextrin, and by forming a complex potentially can prevent or delay the amyloid fibrillation of the insulin and increase the stability of the molecule.

Intradermal injection of icariin-HP-ß-cyclodextrin improved traumatic brain injury via the trigeminal epineurium-brain dura pathway.

The lower bioavailability after oral administration limited icariin applications in central nervous system. Icariin/HP-ß-cyclodextrin (HP-ß-CD) inclusion complex was prepared for acute severe opening traumatic brain injury (TBI) via facial intradermal (i.d.) in the mystacial pad. After fluid percussion-induced TBI, icariin/HP-ß-CD at 0.4 mg/kg i.d. preserved more neurons and oligodendrocytes than intranasal injection (i.n.) or intravenous injection via tail vein (i.v.) and decreased microglia and astrocyte activation. Icariin/HP-ß-CD i.d. reduced apoptosis in cortical penumbra while i.n. and i.v. showed weak or no effects. Icariin/HP-ß-CD i.d. reduced Evans blue leakage and altered CD34, ZO-1, Claudin-5, and beta-catenin expression after TBI. Moreover, icariin/HP-ß-CD promoted human umbilical vein endothelial cells proliferation. Thus, Icariin/HP-ß-CD i.d. improved TBI, including blood-brain barrier opening. Fluorescein 5-isothiocyanate (FITC) and 3,3'-Dioctadecyloxacarbocyanine perchlorate (DiOC18(3)) mimic HP-ß-CD and icariin respectively. FITC and DiOC18(3) were similarly delivered to trigeminal epineurium, perineurium and perivascular spaces or tissues, caudal dura mater, and scattered in trigeminal fasciculus, indicating that icariin/HP-ß-CD was delivered to the brain via trigeminal nerve-dura mater-brain pathways. In sum, intradermal injection in mystacial pad might deliver icariin/HP-ß-CD to the brain and icariin/HP-ß-CD improved acute severe opening TBI.

On the Interactions of Melatonin/ß-Cyclodextrin Inclusion Complex: A Novel Approach Combining Efficient Semiempirical Extended Tight-Binding (xTB) Results with Ab Initio Methods.

Melatonin (MT) is a molecule of paramount importance in all living organisms, due to its presence in many biological activities, such as circadian (sleep-wake cycle) and seasonal rhythms (reproduction, fattening, molting, etc.). Unfortunately, it suffers from poor solubility and, to be used as a drug, an appropriate transport vehicle has to be developed, in order to optimize its release in the human tissues. As a possible drug-delivery system, ß-cyclodextrin (ßCD) represents a promising scaffold which can encapsulate the melatonin, releasing when needed. In this work, we present a computational study supported by experimental IR spectra on inclusion MT/ßCD complexes. The aim is to provide a robust, accurate and, at the same time, low-cost methodology to investigate these inclusion complexes both with static and dynamic simulations, in order to study the main actors that drive the interactions of melatonin with ß-cyclodextrin and, therefore, to understand its release mechanism.

Magnetic dispersive solid-phase extraction of triazole and triazine pesticides from vegetable samples using a hydrophilic-lipophilic sorbent based on maltodextrin- and ß-cyclodextrin-functionalized graphene oxide.

Maltodextrin- and ß-cyclodextrin-functionalized magnetic graphene oxide (mGO/ß-CD/MD), a novel hydrophilic-lipophilic composite, was successfully fabricated and used for the co-extraction of triazines and triazoles from vegetable samples before HPLC-UV analysis. mGO/ß-CD/MD was synthesized by chemical bonding of ß-CD and MD to the surface of mGO, using epichlorohydrin (ECH) as a linker. The successful synthesis of mGO/ß-CD/MD was confirmed by characterization tests, including attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), vibrating sample magnetometry (VSM), thermogravimetric analysis (TGA), energy-dispersive X-ray spectroscopy (EDX), Brunauer-Emmett-Teller (BET), and Barrett-Joyner-Halenda (BJH) analyses. The hydrophobic cavity of ß-CD and a large number of hydroxyl groups on the MD structure contributed to the co-extraction of mentioned pesticides with a wide range of polarity. Under the optimized condition (sorbent amount, 30 mg; desorption time, 10 min; desorption solvent volume, 300 µL; desorption solvent, methanol/acetonitrile (1:1) containing 5% (v/v) acetic acid; extraction time, 20 min; and pH of sample solution, 7.0), good linearity within the range 1.0-1000 µg L-1 (r2 ≥ 0.992) was achieved. Extraction efficiencies were in the range 66.4-95.3%, and the limits of detection were 0.01-0.08 µg L-1. Relative recoveries for spiked samples were obtained in the range 88.4-112.0%, indicating that the matrix effect was insignificant, and good precisions (intra- and inter-day) were also achieved (RSDs < 9.0%, n = 3). The results confirmed that the developed method was efficient for the determination  of trace amounts of pesticides in potato, tomato, and corn samples.

The Interaction of Heptakis (2,6-di-O-Methyl)-ß-cyclodextrin with Mianserin Hydrochloride and Its Influence on the Drug Toxicity.

One tetracyclic antidepressant, mianserin hydrochloride (MIA), has quite significant side effects on a patients' health. Cyclodextrins, which are most commonly used to reduce the undesirable features of contained drugs within their hydrophobic interior, also have the potential to alter the toxic behavior of the drug. The present paper contains investigations and the characteristics of interaction mechanisms for MIA and the heptakis (2,6-di-O-methyl)-ß-cyclodextrin (DM-ß-CD) system, and evaluated the effects of the complexation on MIA cytotoxicity. In order to assess whether there was an interaction between MIA and DM-ß-CD molecules, isothermal titration calorimetry (ITC) have been chosen. Electrospray ionization mass spectrometry (ESI-MS) helped to establish the complex stoichiometry, and circular dichroism spectroscopy was used to describe the process of complex formation. In order to make a wider interpretative perspective, the molecular docking results have been performed. The viability of Chinese hamster cells were investigated in the presence of DM-ß-CD and its complexes with MIA in order to estimate the cytotoxicity of the drug and the conjugate with the chosen cyclodextrin. The viability of B14 cells treated with MIA+DM-ß-CD is lower (the toxicity is higher) than with MIA alone, and no protective effects have been observed for complexes of MIA with DM-ß-CD in any ratio.

Deoxycholic acid induces proinflammatory cytokine production by model oesophageal cells via lipid rafts.

The bile acid component of gastric refluxate has been implicated in inflammation of the oesophagus including conditions such as gastro-oesophageal reflux disease (GORD) and Barrett's Oesophagus (BO). Here we demonstrate that the hydrophobic bile acid, deoxycholic acid (DCA), stimulated the production of IL-6 and IL-8 mRNA and protein in Het-1A, a model of normal oesophageal cells. DCA-induced production of IL-6 and IL-8 was attenuated by pharmacologic inhibition of the Protein Kinase C (PKC), MAP kinase, tyrosine kinase pathways, by the cholesterol sequestering agent, methyl-beta-cyclodextrin (MCD) and by the hydrophilic bile acid, ursodeoxycholic acid (UDCA). The cholesterol-interacting agent, nystatin, which binds cholesterol without removing it from the membrane, synergized with DCA to induce IL-6 and IL-8. This was inhibited by the tyrosine kinase inhibitor genistein. DCA stimulated the phosphorylation of lipid raft component Src tyrosine kinase (Src). while knockdown of caveolin-1 expression using siRNA resulted in a decreased level of IL-8 production in response to DCA. Taken together, these results demonstrate that DCA stimulates IL-6 and IL-8 production in oesophageal cells via lipid raft-associated signaling. Inhibition of this process using cyclodextrins represents a novel therapeutic approach to the treatment of inflammatory diseases of the oesophagus including GORD and BO.

Specific recognition of protein by deep eutectic solvent-based magnetic ß-cyclodextrin molecularly imprinted polymer.

A magnetic ß-cyclodextrin (MCD) surface molecularly imprinted polymer (MIP) based on deep eutectic solvents (DESs) as cross-linker and functional monomer (MCD@DES-MIP) was successfully synthesized for the specific recognition of bovine hemoglobin (BHb). The adsorption behavior of MCD@DES-MIP for BHb was investigated by adsorption thermodynamics, adsorption kinetics, and pH control experiments. The maximum adsorption capacity of MCD@DES-MIP for BHb under the optimized conditions was 195.94 mg g-1 and the imprinting factor was 4.68. In addition, the competitive adsorption experiments demonstrated that MCD@DES-MIP showed excellent selective extraction ability for BHb in the binary mixture of BHb and bovine serum albumin (BSA). The actual sample analysis manifested that MCD@DES-MIP effectively separated BHb from complex samples. The results of circular dichroism spectra proved that the secondary structure of BHb did not change during elution. The result indicated that MCD@DES-MIP can be used as a new imprinting material for the separation and purification of BHb.Graphical abstract Magnetic imprinted microspheres (MCD@DES-MIP) were prepared by free radical polymerization using magnetic ß-cyclodextrin (MCD) as carrier, deep eutectic solvents (DESs) as functional monomer and cross-linker. MCD@DES-MIP show high adsorption capacity and excellent selectivity for BHb.

A cell-free assay implicates a role of sphingomyelin and cholesterol in STING phosphorylation.

Stimulator of interferon genes (STING) is essential for the type I interferon response induced by microbial DNA from virus or self-DNA from mitochondria/nuclei. In response to emergence of such DNAs in the cytosol, STING translocates from the endoplasmic reticulum to the Golgi, and activates TANK-binding kinase 1 (TBK1) at the trans-Golgi network (TGN). Activated TBK1 then phosphorylates STING at Ser365, generating an interferon regulatory factor 3-docking site on STING. How this reaction proceeds specifically at the TGN remains poorly understood. Here we report a cell-free reaction in which endogenous STING is phosphorylated by TBK1. The reaction utilizes microsomal membrane fraction prepared from TBK1-knockout cells and recombinant TBK1. We observed agonist-, TBK1-, "ER-to-Golgi" traffic-, and palmitoylation-dependent phosphorylation of STING at Ser365, mirroring the nature of STING phosphorylation in vivo. Treating the microsomal membrane fraction with sphingomyelinase or methyl-ß-cyclodextrin, an agent to extract cholesterol from membranes, suppressed the phosphorylation of STING by TBK1. Given the enrichment of sphingomyelin and cholesterol in the TGN, these results may provide the molecular basis underlying the specific phosphorylation reaction of STING at the TGN.

Supramolecular Polymerization-Induced Nanoassemblies for Self-Augmented Cascade Chemotherapy and Chemodynamic Therapy of Tumor.

The clinical application of chemodynamic therapy is impeded by the insufficient intracellular H2 O2 level in tumor tissues. Herein, we developed a supramolecular nanoparticle via a simple one-step supramolecular polymerization-induced self-assembly process using platinum (IV) complex-modified ß-cyclodextrin-ferrocene conjugates as supramolecular monomers. The supramolecular nanoparticles could dissociate rapidly upon exposure to endogenous H2 O2 in the tumor and release hydroxyl radicals as well as platinum (IV) prodrugs in situ, which is reduced into cisplatin to significantly promote the generation of H2 O2 in the tumor tissue. Thus, the supramolecular nanomedicine overcomes the limitation of conventional chemodynamic therapy via the self-augmented cascade radical generation and drug release. In addition, dissociated supramolecular nanoparticles could be readily excreted from the body via renal clearance to effectively avoid systemic toxicity and ensure long term biocompatibility of the nanomedicine. This work may provide new insights on the design and development of novel supramolecular nanoassemblies for cascade chemo/chemodynamic therapy.

Rim Binding of Cyclodextrins in Size-Sensitive Guest Recognition.

Cyclodextrins (CDs) are doughnut-shaped cyclic oligosaccharides having a cavity and two rims. Inclusion binding in the cavity has long served as a classic model of molecular recognition, and rim binding has been neglected. We found that CDs recognize guests by size-sensitive binding using the two rims in addition to the cavity, using single-molecule electron microscopy and a library of graphitic cones as a solid-state substrate for complexation. For example, with its cavity and rim binding ability combined, γ-CD can recognize a guest of radius between 4 and 9 Å with a size-recognition precision of better than 1 Å, as shown by structural analysis of thousands of individual specimens and statistical analysis of the data thereof. A 2.5 ms resolution electron microscopic video provided direct evidence of the process of size recognition. The data suggest the occurrence of the rim binding mode for guests larger than the size of the CD cavity and illustrate a unique application of dynamic molecular electron microscopy for deciphering the spatiotemporal details of supramolecular events.

Shear-thinning and self-healing nanohybrid alginate-graphene oxide hydrogel based on guest-host assembly.

The study aims to develop a novel nanohybrid shear-thinning hydrogel with fast gelation, and variable mechanical and biological properties. This nanohybrid hydrogel was developed via self-assembly guest-host interaction between ß-cyclodextrin modified alginate (host macromere, Alg-CD) and adamantine modified graphene oxide (guest macromere, Ad-GO) and subsequent ionic crosslinking process. We found that the rheological and mechanical properties of hydrogels were controlled via macromere concentration and the host: guest macromere ratio, due to the modulation of crosslinking density and network structure. Noticeably, 12%(1:2) dual-crosslinked hydrogel (2DC12) significantly improved the strength (1.3-folds) and toughness compared to 10%(1:4) dual-crosslinked hydrogel (4DC10). Furthermore, the hydrogel erosion and cytocompatibility relied on the designed parameters. Remarkably, 2DC12 showed less than 20% weight loss after 20 days of incubation in physiological solution and more than 200% cell survival after five days. In conclusion, the nanohybrid Alg-GO hydrogel could be used as an injectable hydrogel for soft tissue engineering applications.

The Viability and Anti-Inflammatory Effects of Hyaluronic Acid-Chitlac-Tracimolone Acetonide- ß-Cyclodextrin Complex on Human Chondrocytes.

OBJECTIVE: To compare the effects of the complex triamcinolone acetonide-hydroxypropyl-ß-cyclodextrin (TA-CD) on in vitro inflamed primary human articular chondrocytes in the presence or absence of the mixture hyaluronic acid-Chitlac, a lactose-modified chitosan (HA-CTL). DESIGN: Changes in cell viability and pro-inflammatory cytokines gene expression were analyzed in human chondrocytes using an in vitro model of macrophage-mediated inflammation. Human monocytes U937 were differentiated to macrophages by phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharides (LPS). The anti-inflammatory effects of the complex TA-CD and HA-CTL mixture were assessed on chondrocytes exposed for 24 hours to U937 conditioned medium (CM), by quantitative polymerase chain reaction analysis. RESULTS: The TA-CD viability was enhanced by the presence of the HA-CTL mixture in chondrocyte cultures. The exposure of cells to CM significantly increased interleukin-1ß and interleukin-6 gene expression, and when the complex TA-CD was added to the inflamed cells, gene transcription of cytokines was restored to near baseline values, both in the presence or in the absence of HA-CTL mixture. CONCLUSION: The addition of HA-CTL mixture significantly attenuated cytotoxicity induced by TA and preserved the anti-inflammatory effects, thus confirming the chondroprotective role of the HA-CTL mixture.

Experimental characterization of the association of ß-cyclodextrin and eight novel cyclodextrin derivatives with two guest compounds.

We investigate the binding of native ß-cyclodextrin (ß-CD) and eight novel ß-CD derivatives with two different guest compounds, using isothermal calorimetry and 2D NOESY NMR. In all cases, the stoichiometry is 1:1 and binding is exothermic. Overall, modifications at the 3' position of ß-CD, which is at the secondary face, weaken binding by several kJ/mol relative to native ß-CD, while modifications at the 6' position (primary face) maintain or somewhat reduce the binding affinity. The variations in binding enthalpy are larger than the variations in binding free energy, so entropy-enthalpy compensation is observed. Characterization of the bound conformations with NOESY NMR shows that the polar groups of the guests may be situated at either face, depending on the host molecule, and, in some cases, both orientations are populated. The present results were used in the SAMPL7 blinded prediction challenge whose results are detailed in the same special issue of JCAMD.

Isolation of Lipid Rafts (Detergent-Resistant Microdomains) and Comparison to Extracellular Vesicles (Exosomes).

Lipid rafts (LRs) represent cellular microdomains enriched in sphingolipids and cholesterol which may fuse to form platforms in which signaling molecules can be organized and regulated (Simons and Ikonen, Nature 387:569-572, 1997; Pike, Biochem J 378:281-292, 2004; Grassme et al., J Immunol 168: 300-307, 2002; Cheng et al., J Exp Med 190:1549-1550, 1999; Kilkus et al., J Neurosci Res 72(1) 62-75, 2003). In a proposed Model 1 (Cheng et al., J Exp Med 190:1549-1550, 1999) the LR has a well-ordered central core composed mainly of cholesterol and sphingolipids that is surrounded by a zone of decreasing lipid order. Detergents such as Triton X-100 can solubilize the core (and a significant amount of phosphoglyceride), but the LRs will be insoluble at 4 °C and be enriched in a well-characterized set of biomarkers. Model 2 proposes that the LRs are homogeneous, but there is selectivity in the lipids (and proteins) extracted by the 1% Triton X-100. Model 3 proposes LRs with distinct lipid compositions are highly structured and can be destroyed by binding molecules such as beta-methylcyclodextrin or filipin. These may be Caveolin in some cell types but not in brain. Since it is unlikely that two LR preparations will be exactly the same this review will concentrate on LRs defined as "small (50 nm) membranous particles which are insoluble in 1% Triton X-100 at 4 °C and have a low buoyant density (Simons and Ikonen, Nature 387:569-572, 1997; Pike, Biochem J 378:281-292, 2004; Grassme et al., J Immunol 168: 300-307, 2002; Cheng et al., J Exp Med 190:1549-1550, 1999; Kilkus et al., J Neurosci Res 72(1):62-75, 2003; Testai et al., J Neurochem 89:636-644, 2004). We will present a generic method for isolating LRs for both lipidomic, proteomic, and cellular signaling analysis [1-6].