Trehalose-based neuroprotective autophagy inducers.

A small set of trehalose-centered putative autophagy inducers was rationally designed and synthesized, with the aim to identify more potent and bioavailable autophagy inducers than free trehalose, and to acquire information about their molecular mechanism of action. Several robust, high yield routes to key trehalose intermediates and small molecule prodrugs (2-5), putative probes (6-10) and inorganic nanovectors (12a - thiol-PEG-triazole-trehalose constructs 11) were successfully executed, and compounds were tested for their autophagy-inducing properties. While small molecules 2-11 showed no pro-autophagic behavior at sub-millimolar concentrations, trehalose-bearing PEG-AuNPs 12a caused measurable autophagy induction at an estimated 40 µM trehalose concentration without any significant toxicity at the same concentration.

Analysis of In Vitro Cytotoxicity of Carbohydrate-Based Materials Used for Dissolvable Microneedle Arrays.

PURPOSE: Dissolvable microneedle arrays (MNAs) can be used to realize enhanced transdermal and intradermal drug delivery. Dissolvable MNAs are fabricated from biocompatible and water-soluble base polymers, and the biocargo to be delivered is integrated with the base polymer when forming the MNAs. The base polymer is selected to provide mechanical strength, desired dissolution characteristics, and compatibility with the biocargo. However, to satisfy regulatory requirements and be utilized in clinical applications, cytotoxicity of the base polymers should also be thoroughly characterized. This study systematically investigated the cytotoxicity of several important carbohydrate-based base polymers used for production of MNAs, including carboxymethyl cellulose (CMC), maltodextrin (MD), trehalose (Treh), glucose (Gluc), and hyaluronic acid (HA). METHODS: Each material was evaluated using in vitro cell-culture methods on relevant mouse and human cells, including MPEK-BL6 mouse keratinocytes, NIH-3T3 mouse fibroblasts, HaCaT human keratinocytes, and NHDF human fibroblasts. A common laboratory cell line, human embryonic kidney cells HEK-293, was also used to allow comparisons to various cytotoxicity studies in the literature. Dissolvable MNA materials were evaluated at concentrations ranging from 3 mg/mL to 80 mg/mL. RESULTS: Qualitative and quantitative analyses of cytotoxicity were performed using optical microscopy, confocal fluorescence microscopy, and flow cytometry-based assays for cell morphology, viability, necrosis and apoptosis. Results from different methods consistently demonstrated negligible in vitro cytotoxicity of carboxymethyl cellulose, maltodextrin, trehalose and hyaluronic acid. Glucose was observed to be toxic to cells at concentrations higher than 50 mg/mL. CONCLUSIONS: It is concluded that CMC, MD, Treh, HA, and glucose (at low concentrations) do not pose challenges in terms of cytotoxicity, and thus, are good candidates as MNA materials for creating clinically-relevant and well-tolerated biodissolvable MNAs.

Primary study on the toxic mechanism of vanadyl trehalose in Kunming mice.

It has been shown that vanadyl trehalose could lower blood glucose but show mild toxicity to the stomach and intestine in diabetic Kunming mice. We analysed antioxidant levels, pro-inflammatory cytokine expression, apoptosis factors and intestinal microflora alteration to explore the mechanism of vanadyl trehalose toxicity in Kunming mice. The results revealed that oral administration of vanadyl trehalose at tested dose caused significant changes in oxidative stress factor (MDA levels elevated but SOD and T-AOC decreased), expression of inflammatory factor (IL-1ß, COX-2, TNF-α and iNOS increased), and apoptosis factor (Bcl-2/Bax decreased and caspase-3 increased), and intestinal microflora dysbiosis (the number of Enterobacteriaceae and Enterococcus increased and Lactobacillus and Bifidobacterium decreased) relative to the control of Kunming mice. These results suggest that the toxic mechanisms of vanadyl trehalose on the stomach and intestine likely involve activation of the oxidative stress system, increased inflammatory response, promotion of apoptosis and the disruption of the normal intestinal microflora.

Improvement of post-thaw sperm survivals using liquid nitrogen vapor in a spermcasting oyster Ostrea angasi.

Low survival of cryopreserved sperm impedes the application of cryopreservation technique in spermcasting oyster species. This study developed a simple method of liquid nitrogen vapor freezing to improve post-thaw sperm survival in the spermcasting oyster Ostrea angasi. The results indicate that the permeable cryoprotectants, dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) were non-toxic to sperm up to 20% concentration and 90 min exposure whereas methanol at 10% or higher was toxic to sperm for any exposure over 30 min. Among the treatments with permeable cryoprotectants, 15% EG produced the highest post-thaw sperm motility. Sperm motility was further improved by the addition of non-permeable cryoprotectants (trehalose and glucose), with 15% EG + 0.2 M trehalose resulting in the highest post-thaw sperm motility among all the combinations evaluated. The durations of 20, 30 and 60 min equilibrations produced a higher post-thaw sperm motility and plasma membrane integrity (PMI) than 10 min. Higher post-thaw motility and PMI were achieved by freezing sperm at the 8 cm height from the liquid nitrogen surface than at the 2, 4, 6, 10 or 12 cm height. Holding sperm for 10 min in liquid nitrogen vapor produced higher post-thaw motility and PMI than for 2, 5 or 20 min. The cryopreservation protocol developed in this study improved both post-thaw motility and PMI of O. angasi sperm at least 15% higher than those cryopreserved using programmable freezing method. Liquid nitrogen vapor freezing might have greater applicability in improving post-thaw sperm quality of spermcasting oyster species.

Acute toxicity, twenty-eight days repeated dose toxicity and genotoxicity of vanadyl trehalose in kunming mice.

A new trend has been developed using vanadium and organic ligands to form novel compounds in order to improve the beneficial actions and reduce the toxicity of vanadium compounds. In present study, vanadyl trehalose was explored the oral acute toxicity, 28 days repeated dose toxicity and genotoxicity in Kunming mice. The Median Lethal Dose (LD50) of vanadyl trehalose was revealed to be 1000 mg/kg body weight in fasted Kunming mice. Stomach and intestine were demonstrated to be the main target organs of vanadyl trehalose through 28 days repeated dose toxicity study. And vanadyl trehalose also showed particular genotoxicity through mouse bone marrow micronucleus and mouse sperm malformation assay. In brief, vanadyl trehalose presented certain, but finite toxicity, which may provide experimental basis for the clinical application.

Nanoparticle-mediated intracellular delivery enables cryopreservation of human adipose-derived stem cells using trehalose as the sole cryoprotectant.

In this study, pH responsive genipin-cross-linked Pluronic F127-chitosan nanoparticles (GNPs) was synthesized to encapsulate trehalose for intracellular delivery to cryopreserve primary human adipose-derived stem cells (hADSCs). Trehalose is a disaccharide of glucose used by lower organisms to survive extreme cold in nature and has been used to cryopreserve various biomacromolecules. However, it does not enter mammalian cells because of its highly hydrophilic nature, and has only been used in combination with other cell-penetrating cryoprotectants (such as dimethyl sulfoxide, DMSO) to cryopreserve mammalian cells. Our data show that trehalose can be efficiently encapsulated in our GNPs for intracellular delivery, which enables cryopreservation of primary hADSCs using the nontoxic sugar as the sole cryoprotectant. This capability is important because the conventional approach of cryopreserving mammalian cells using highly toxic (at body temperature) cell-penetrating cryoprotectants requires multistep washing of the cryopreserved cells to remove the toxic cryoprotectant for further use, which is time-consuming and associated with significant cell loss (∼10% during each washing step). By contrast, the trehalose-cryopreserved cells can be used without washing, which should greatly facilitate the wide application of the burgeoning cell-based medicine.

Multiple toxicity studies of trehalose in mice by intragastric administration.

In the present study, aberration, body weight, food consumption, haematology, organ coefficients, and both gross and microscopic appearance of some histiocytes were compared between the test and control groups. A sperm abnormality test, bone marrow cell micronucleus test, and a haematology study were conducted at levels of 1.25 g/kg, 2.5 g/kg, and 5 g/kg of trehalose. In both the sperm abnormality test and bone marrow cell micronucleus test, statistically significant differences were observed between the positive control and treatment groups (P<0.05), while no statistical difference was observed among the negative control, high dose, moderate dose and low dose groups (P>0.05). In the haematology study, there was no significant difference found from the controls at P>0.05. The results obtained in the present study could support the conclusion that consumption of trehalose has no adverse effects for humans.

Cryopreservation of Greenshell™ mussel (Perna canaliculus) trochophore larvae.

The Greenshell™ mussel (Perna canaliculus) is the main shellfish species farmed in New Zealand. The aim of this study was to evaluate the effects of cryoprotectant concentration, loading and unloading strategy as well as freezing and thawing method in order to develop a protocol for cryopreservation of trochophore larvae (16-20 h old). Toxicity tests showed that levels of 10-15% ethylene glycol (EG) were not toxic to larvae and could be loaded and unloaded in a single step. Through cryopreservation experiments, we designed a cryopreservation protocol that enabled 40-60% of trochophores to develop to D-larvae when normalized to controls. The protocol involved: holding at 0 °C for 5 min, then cooling at 1 °C min⁻¹ to -10 °C, holding for a further 5 min, then cooling at 0.5 °C min⁻¹ to -35 °C followed by a 5 min hold and then plunging into liquid nitrogen. A final larval rearing experiment of 18 days was conducted to assess the ability of these frozen larvae to develop further. Results showed that only 2.8% of the frozen trochophores were able to develop to competent pediveligers.

The physicochemical properties and chemical composition of trehalose lipids produced by Rhodococcus erythropolis 51T7.

This study analyzed the chemical and physical properties of a biosurfactant synthesized by Rhodococcus sp. 51T7. The biosurfactant was a trehalose tetraester (THL) consisting of six components: one major and five minor. The hydrophobic moieties ranged in size from 9 to 11 carbons. The critical micelle concentration (CMC) was 0.037g L(-1) and the interfacial tension against hexadecane was 5mN m(-1). At pH 7.4 the glycolipid CMC/critical aggregation concentration (CAC) was 0.05g L(-1) and at pH 4 it was 0.034g L(-1). A phase diagram revealed effective emulsification with water and paraffin or isopropyl myristate. A composition of 11.3-7.5-81.8 (isopropyl myristate-THL-W) was stable for at least 3 months. The HLB was 11 and the phase behaviour of the glycolipid revealed the formation of lamellar and hexagonal liquid-crystalline textures.

Stage-dependent hatching responses of rohu (Labeo rohita) embryos to different concentrations of cryoprotectants and temperatures.

Hatching performances of three embryonic stages of postfertilization rohu (Labeo rohita) (9-, 12-, and 15-h) were examined after treatment with various concentrations (0.5-4.5M) of two cryoprotectants (methanol and propylene glycol) supplemented with 0.1M trehalose. Different lengths of storage (1-48 h) and temperature (-4 degrees C to ambient) were studied. Of the three stages of embryonic development, the 12-h stage proved to be the most suitable stage for low temperature storage, showing the highest percentage of hatch out (72+/-2%) with 2.0M methanol and 0.1M trehalose. Methanol was more useful for storage at higher temperatures and propylene glycol at subzero temperatures. The maximum possible duration of effective storage of 12-h embryos was 31h in 2.0M methanol at 0 degrees C. No hatch out was found beyond 31h of storage with all concentrations of methanol at 0 degrees C. The results of interactions was that the optimal concentration of methanol was 3.0M at 4 degrees C, 2.0M at 0 degrees C, and 1.5M at 4 degrees C. Among three embryonic stages 12-h stage showed better results in trehalose treatment than sucrose. Among all concentrations of trehalose tested 0.1M gave the maximal survival rate of the rohu embryos.

In vivo administration of mycobacterial cord factor (Trehalose 6, 6'-dimycolate) can induce lung and liver granulomas and thymic atrophy in rabbits.

Trehalose 6,6'-dimycolate (TDM) is a cell surface molecule of Mycobacterium tuberculosis. TDM induced a loss of body weight and prominent granulomas in the liver and lungs by the intravenous injection of TDM into rabbits. TDM also induced atrophy of the thymus and spleen due to apoptosis. By contrast, sulfolipid (2,3,6, 6'-tetraacyl trehalose 2'-sulfate) induced neither toxicity, nor granuloma formation, nor atrophy of the thymus and spleen. In rabbits the histopathological changes were more dramatic than in mice. The rabbit model may be more sensitive and may provide more information on the beneficial or pathological effects of TDM.

The optimal equilibration time for mouse embryos frozen by vitrification with trehalose.

To determine the best equilibration time in the cryoprotectant before rapid cooling, 8-cell mouse embryos were exposed to a vitrification solution containing ethylene glycol, Ficoll and trehalose in modified phosphate-buffered saline at 5 degrees C for varying periods of time. They were frozen using an ultra rapid freezing method, thawed in a 20 degrees C water bath and cultured for 24 h with 5-bromo-2-deoxyuridine. Embryo development and the number of sister chromatid exchanges, a sensitive indicator of genetic damage, were observed. The results demonstrated that embryo development after freezing and thawing was similar among the groups exposed for periods of 5-40 min. However, the number of sister chromatid exchanges was significantly smaller in the group exposed for 5 min, indicating that this was the safest equilibration time in the vitrification solution.

Synergistic action of cord factor and mycobacterial sulfatides on mitochondria.

The mechanism of a synergistic toxicity of 6,6'-dimycoloyl-alpha,alpha'-d-trehalose (cord factor) and 2,3,6,6'-tetraacyl-alpha,alpha'-d-trehalose 2'-sulfate (sulfolipid I) for mice was studied. Sulfolipid I was entirely nontoxic, but it markedly accelerated the lethal toxicity of cord factor for mice. In vivo, sulfolipid I affected neither respiration nor accompanying phosphorylation of mouse liver mitochondria, whereas in vitro, it induced a swelling and disruption of mitochondrial membranes and strongly inhibited mitochondrial oxidative phosphorylation. The effect of sulfolipid I on mitochondrial structure and function in vitro was neutralized by bovine serum albumin and various animal sera, whereas that of cord factor and cord factor plus sulfolipid I was not prevented by bovine serum albumin. The simultaneous injection of cord factor and sulfolipid I caused an intensive fragmentation of mitochondria and a marked decrease in respiratory and phosphorylative activity in mitochondria. These data indicate that sulfolipid I can achieve an effective attack on mitochondria in combination with cord factor in vivo and induces heavier damage in mitochondrial structure and function than that produced by cord factor alone.

Isolation and biochemical activities of trehalose-6-monomycolate of Mycobacterium tuberculosis.

A monoester of trehalose linked at the 6-position with mycolic acids (trehalose-6-monomycolate) was isolated from the wax D fraction of virulent human Mycobacterium tuberculosis, and its biochemical action on host-cell mitochondria was studied. Trehalose-6-monomycolate showed a delayed toxicity for mice. The 50% lethal dose at 2 weeks was 452 mug. It induced in vitro a swelling of mouse liver mitochondria and uncoupled respiration and phosphorylation in the nicotinamide adenine dinucleotide pathway of the electron transport chain. The site of functional damage was located specifically at coupling site II. Mitochondrial adenosine triphosphatase was slightly stimulated by trehalose-6-monomycolate. These findings indicate that trehalose-6-monomycolate affects mitochondrial oxidative phosphorylation in a similar manner to, but to a lesser extent than, trehalose-6, 6'-dimycolate (cord factor) of M. tuberculosis.