Novel PEGylated derivatives of α-tocopherol for nanocarrier formulations; synthesis, characterization and in vitro cytotoxicity against MCF-7 breast cancer cells.

Despite numerous beneficial therapeutic effects namely antioxidant and anti-inflammatory activity, Vitamin E has limited clinical applications due to its low water solubility. Throughout the present work, α-tocopherol's new PEGylated derivatives alongside with polyethylene glycol 300 (α-1TPGT300), 400 (α-TPGT400), and 1000 (α-TPGT1000) were synthesized. A 1,2,3-triazole ring was utilized as a linker for the attachment of alpha tocopherol to the PEGs through a click reaction. The purified derivatives were characterized by the means of 1H NMR, 13C NMR, mass spectroscopy, UV-vis and FT-IR methods. Synthesized derivatives' capacity to produce self-assembly nanoparticles was evaluated employing the critical micelle concentration (CMC) values. The stability of the micelles was studied by size analysis. In vitro cytotoxicity of the products was investigated using MTT assay against MCF-7 breast cancer cells. The IC50 value for TPGT1000 after 24 h treatment was 15.0 ± 1.8 µM, whereas no significant cytotoxicity effect was observed following the treatment of MCF-7 cells by TPGT300, 400. The present study showed that polymeric micelle TPGT1000 possessed better physicochemical and biological properties including relatively lower CMC value, higher stability in FBS environment in addition to higher cytotoxicity against MCF-7 breast cancer cells compared to the lower molecular weight PEGylated derivatives. These results confirmed that increasing PEG chain length left a positive effect on the polymeric micelle properties and also improved the cytotoxicity effect of new PEGylated vitamin E derivatives.

Synthetic α-Tocopherol, Compared with Natural α-Tocopherol, Downregulates Myelin Genes in Cerebella of Adolescent Ttpa-null Mice.

BACKGROUND: Vitamin E (α-tocopherol; α-T) deficiency causes spinocerebellar ataxia. α-T supplementation improves neurological symptoms, but little is known about the differential bioactivities of natural versus synthetic α-T during early life. OBJECTIVE: We assessed the effects of dietary α-T dose and source on tissue α-T accumulation and gene expression in adolescent α-tocopherol transfer protein-null (Ttpa-/-) mice. METHODS: Three-week-old male Ttpa-/- mice (n  = 7/group) were fed 1 of 4 AIN-93G-based diets for 4 wk: vitamin E deficient (VED; below α-T limit of detection); natural α-T, 600 mg/kg diet (NAT); synthetic α-T, 816 mg/kg diet (SYN); or high synthetic α-T, 1200 mg/kg diet (HSYN). Male Ttpa+/+ littermates fed AIN-93G [75 mg synthetic α-T (CON)] served as controls (n  = 7). At 7 wk of age, tissue α-T concentrations and stereoisomer profiles were measured for all groups. RNA-sequencing was performed on cerebella of Ttpa-/- groups. RESULTS: Ttpa-/- mice fed VED had undetectable brain α-T concentrations. Cerebral cortex α-T concentrations were greater in Ttpa-/- mice fed NAT (9.1 ± 0.7 nmol/g), SYN (10.8 ± 1.0 nmol/g), and HSYN (13.9 ± 1.6 nmol/g) compared with the VED group but were significantly lower than in Ttpa+/+ mice fed CON (24.6 ± 1.2 nmol/g) (P < 0.001). RRR-α-T was the predominant stereoisomer in brains of Ttpa+/+ mice (∼40%) and Ttpa-/- mice fed NAT (∼94%). α-T stereoisomer composition was similar in brains of Ttpa-/- mice fed SYN and HSYN (2R: ∼53%; 2S: ∼47%). Very few of the 16,774 genes measured were differentially expressed. However, compared with the NAT diet, HSYN significantly downregulated 20 myelin genes, including 2 transcription factors: SRY-box transcription factor 10 (Sox10) and myelin regulatory factor (Myrf), and several downstream target genes (false discovery rate <0.05). CONCLUSIONS: High-dose synthetic α-T compared with natural α-T alters myelin gene expression in the adolescent mouse cerebellum, which could lead to morphological and functional abnormalities later in life.

Vitamin E succinate-grafted-chitosan/chitosan oligosaccharide mixed micelles loaded with C-DMSA for Hg2+ detection and detoxification in rat liver.

AIM: To determine whether the use of a mixed polymeric micelle delivery system based on vitamin E succinate (VES)-grafted-chitosan oligosaccharide (CSO)/VES-grafted-chitosan (CS) mixed micelles (VES-g-CSO/VES-g-CS MM) enhances the delivery of C-DMSA, a theranostic fluorescent probe, for Hg2+ detection and detoxification in vitro and in vivo. METHODS: Mixed micelles self-assembled from two polymers, VES-g-CSO and VES-g-CS, were used to load C-DMSA and afforded C-DMSA@VES-g-CSO/VES-g-CS MM for cell and in vivo applications. Fluorescence microscopy was used to assess C-DMSA cellular uptake and Hg2+ detection in L929 cells. C-DMSA@VES-g-CSO/VES-g-CS MM was then administered intravenously. Hg2+ detection was assessed by fluorescence microscopy in terms of bio-distribution while detoxification efficacy in Hg2+-poisoned rat models was evaluated in terms of mercury contents in blood and in liver. RESULTS: The C-DMSA loaded mixed micelles, C-DMSA@VES-g-CSO/VES-g-CS MM, significantly enhanced cellular uptake and detoxification efficacy of C-DMSA in Hg2+ pretreated human L929 cells. Evidence from the reduction of liver coefficient, mercury contents in liver and blood, alanine transaminase and aspartate transaminase activities in Hg2+ poisoned SD rats treated with the mixed micelles strongly supported that the micelles were effective for Hg2+ detoxification in vivo. Furthermore, ex vivo fluorescence imaging experiments also supported enhanced Hg2+ detection in rat liver. CONCLUSION: The mixed polymeric micelle delivery system could significantly enhance cell uptake and efficacy of a theranostic probe for Hg2+ detection and detoxification treatment in vitro and in vivo. Moreover, this nanoparticle drug delivery system could achieve targeted detection and detoxification in liver.

Synthesis of Daidzein Glycosides, α-Tocopherol Glycosides, Hesperetin Glycosides by Bioconversion and Their Potential for Anti-Allergic Functional-Foods and Cosmetics.

Daidzein is a common isoflavone, having multiple biological effects such as anti-inflammation, anti-allergy, and anti-aging. α-Tocopherol is the tocopherol isoform with the highest vitamin E activity including anti-allergic activity and anti-cancer activity. Hesperetin is a flavone, which shows potent anti-inflammatory effects. These compounds have shortcomings, i.e., water-insolubility and poor absorption after oral administration. The glycosylation of bioactive compounds can enhance their water-solubility, physicochemical stability, intestinal absorption, and biological half-life, and improve their bio- and pharmacological properties. They were transformed by cultured Nicotiana tabacum cells to 7-ß-glucoside and 7-ß-gentiobioside of daidzein, and 3'- and 7-ß-glucosides, 3',7-ß-diglucoside, and 7-ß-gentiobioside of hesperetin. Daidzein and α-tocopherol were glycosylated by galactosylation with ß-glucosidase to give 4'- and 7-ß-galactosides of daidzein, which were new compounds, and α-tocopherol 6-ß-galactoside. These nine glycosides showed higher anti-allergic activity, i.e., inhibitory activity toward histamine release from rat peritoneal mast cells, than their respective aglycones. In addition, these glycosides showed higher tyrosinase inhibitory activity than the corresponding aglycones. Glycosylation of daidzein, α-tocopherol, and hesperetin greatly improved their biological activities.

α-Tocopherol-ascorbic acid hybrid antioxidant based cationic amphiphile for gene delivery: Design, synthesis and transfection.

Natural antioxidants and vitamins have potential to protect biological systems from peroxidative damage induced by peroxyl radicals, α-tocopherol (Vitamin E, lipid soluble) and ascorbic acid (vitamin C, water soluble), well known natural antioxidant molecules. In the present study we described the synthesis and biological evaluation of hybrid of these two natural antioxidants with each other via ammonium di-ethylether linker, Toc-As in gene delivery. Two control cationic lipids N14-As and Toc-NOH are designed in such a way that one is with ascorbic acid moiety and no tocopherol moiety; another is with tocopherol moiety and no ascorbic acid moiety respectively. All the three cationic lipids can form self-assembled aggregates. The antioxidant efficiencies of the three lipids were compared with free ascorbic acid. The cationic lipids (Toc-As, N14-As and Toc-NOH) were formulated individually with a well-known fusogenic co-lipid DOPE and characterization studies such as DNA binding, heparin displacement, size, charge, circular dichroism were performed. The biological characterization studies such as cell viability assay and in vitro transfection studies were carried out with the above formulations in HepG2, Neuro-2a, CHO andHEK-293T cell lines. The three formulations showed their transfection efficiencies with highest in Toc-As, moderate inN14-As and least in Toc-NOH. Interestingly, the transfection efficiency observed with the antioxidant based conjugated lipid Toc-As is found to be approximately two and half fold higher than the commercially available lipofectamine 2000 at 4:1 charge ratio in Hep G2 cell lines. In the other cell lines studied the efficiency of Toc-As is found to be either higher or similarly active compared to lipofectamine 2000. The physicochemical characterization results show that Toc-As lipid is showing maximum antioxidant potency, strong binding with pDNA, least size and optimal zeta potential. It is also found to be least toxic in all the cell lines studied especially in Neuro-2a cell lines when compared to other two lipids. In summary, the designed antioxidant lipid can be exploited as a delivering system for treating ROS related diseases such as malignancy, brain stroke, etc.

Synthesis of 2,2-Dialkyl Chromanes by Intramolecular Ullmann C-O Coupling Reactions toward the Total Synthesis of D-α-Tocopherol.

The complete synthesis of D-α-tocopherol was achieved using our developed-Ullmann C-O coupling reaction as a key reaction. The synthesis of the core structure of D-α-tocopherol, which is a chiral chromane, has never been reported using intramolecular Ullmann C-O coupling reactions owing to the low reactivity of electron-rich iodoarenes with tertiary alcohols. Because the developed intramolecular C-O coupling reactions prefer electron-rich iodoarenes with tertiary alcohols, we successfully synthesized the chiral chromane core and achieved the total synthesis of D-α-tocopherol.

Polymeric Micelles Based on Modified Glycol Chitosan for Paclitaxel Delivery: Preparation, Characterization and Evaluation.

Amphiphilic polymer of α-tocopherol succinate modified glycol chitosan (TS-GC) was successfully constructed by conjugating α-tocopherol succinate to the skeleton of glycol chitosan and characterized by Fourier-transform infrared (FT-IR) and proton nuclear magnetic resonance (¹H-NMR). In aqueous milieu, the conjugates self-assembled to micelles with the critical aggregation concentration of 7.2 × 10−3 mg/mL. Transmission electron microscope (TEM) observation and dynamic light scattering (DLS) measurements were carried out to determine the physicochemical properties of the micelles. The results revealed that paclitaxel (PTX)-loaded TS-GC micelles were spherical in shape. Moreover, the PTX-loaded micelles showed increased particle sizes (35 nm vs. 142 nm) and a little reduced zeta potential (+19 mV vs. +16 mV) compared with blank micelles. The X-ray diffraction (XRD) spectra demonstrated that PTX existed inside the micelles in amorphous or molecular state. In vitro and in vivo tests showed that the PTX-loaded TS-GC micelles had advantages over the Cremophor EL-based formulation in terms of low toxicity level and increased dose, which suggested the potential of the polymer as carriers for PTX to improve their delivery properties.

pH-sensitive inulin-based nanomicelles for intestinal site-specific and controlled release of celecoxib.

Aiming at a site-specific drug release in the lower intestinal tract, this paper deals with the synthesis and physicochemical/biological characterization of pH-sensitive nanomicelles from an inulin (INU) amphiphilic derivative. To allow an intestinal site specific release of the payload, INU-Vitamin E (INVITE) bioconjugates were functionalized with succinic anhydride to provide the system with pH-sensitive groups preventing a premature release of the payload into the stomach. The obtained INVITESA micelles resulted nanosized, with a low critical aggregation concentration and the release studies showed a marked pH-dependent release. The drug loading stabilized the micelles against the acidic hydrolysis. From transport studies on Caco-2 cells, resulted that INVITESA nanomicelles cross the cellular monolayer but are actively re-transported in the secretory (basolateral-apical) direction when loaded in apical side. It suggests that the entrapped drug could not be absorbed before the release from the micelles, enabling so a local release of the active.

Highly stereoselective construction of the C2 stereocentre of α-tocopherol (vitamin E) by asymmetric addition of Grignard reagents to ketones.

Tertiary alcohol precursors of both C2 diastereoisomers of α-tocopherol were prepared in three ways by our recently reported asymmetric Grignard synthesis. The versatility of Grignard chemistry inherent in its three-way disconnection was exploited to allow the synthesis of three product grades: 77 : 23 dr (5 steps), 81 : 19 dr (5 steps) and 96 : 4 dr (7 steps, one gram scale) from readily available and abundant starting materials. The products were converted to their respective α-tocopherols in 3 steps, which allowed a definitive re-assignment of their absolute configurations.

Optimization of olive oil based O/W nanoemulsions prepared through ultrasonic homogenization: A response surface methodology approach.

The present study was conducted to prepare co-surfactant free, olive-oil based alpha tocopherol nanoemulsions, using a food grade non-ionic surfactant. Response surface methodology (RSM) was used to determine the effects of independent variables (ultrasonic homogenization time, olive oil concentrations and surfactant contents) on different physico-chemical characteristics of O/W nanoemulsions. This study was carried out using a central composite design. The coefficients of determination were greater than 0.900 for all response variables and there were significant effects of independent variables on all responses. The optimum levels of independent variables for the preparation of nanoemulsions were 3min. ultrasonic homogenization time, 4% olive oil content and 2.08% surfactant concentration. The physico-chemical responses at these levels were 151.68nm particle size, 7.17% p-anisidine and 88.64% antioxidant activity. These results will help in design of nanoemulsions with optimum independent variables.

Succinate ester derivative of δ-tocopherol enhances the protective effects against 60Co γ-ray-induced hematopoietic injury through granulocyte colony-stimulating factor induction in mice.

α-tocopherol succinate (α-TOS), γ-tocotrienol (GT3) and δ-tocotrienol (DT3) have drawn large attention due to their efficacy as radioprotective agents. α-TOS has been shown to act superior to α-tocopherol (α-TOH) in mice by reducing lethality following total body irradiation (TBI). Because α-TOS has been shown to act superior to α-tocopherol (α-TOH) in mice by reducing lethality following total body irradiation (TBI), we hypothesized succinate may be contribute to the radioprotection of α-TOS. To study the contributions of succinate and to identify stronger radioprotective agents, we synthesized α-, γ- and δ-TOS. Then, we evaluated their radioprotective effects and researched further mechanism of δ-TOS on hematological recovery post-irradiation. Our results demonstrated that the chemical group of succinate enhanced the effects of α-, γ- and δ-TOS upon radioprotection and granulocyte colony-stimulating factor (G-CSF) induction, and found δ-TOS a higher radioprotective efficacy at a lower dosage. We further found that treatment with δ-TOS ameliorated radiation-induced pancytopenia, augmenting cellular recovery in bone marrow and the colony forming ability of bone marrow cells in sublethal irradiated mice, thus promoting hematopoietic stem and progenitor cell recovery following irradiation exposure. δ-TOS appears to be an attractive radiation countermeasure without known toxicity, but further exploratory efficacy studies are still required.

Distinction of synthetic dl-α-tocopherol from natural vitamin E (d-α-tocopherol) by reversed-phase liquid chromatography. Enhanced selectivity of a polymeric C18 stationary phase at low temperature and/or at high pressure.

Separation of diastereomers of dl-α-tocopherol was studied by reversed-phase liquid chromatography using three types of stationary phases, polymeric ODS, polymeric C30, and monomeric ODS. Polymeric ODS stationary phase (Inertsil ODS-P, 3mmID, 20cm) was effective for the separation of the isomers created by the presence of three chiral centers on the alkyl chain of synthetic dl-α-tocopherol. Considerable improvement of the separation of isomers was observed on ODS-P phase at high pressure and at low temperature. Complete separation of four pairs of diastereomers was achieved at 12.0°C, 536bar, while three peaks were observed when the separation was carried out either at 12.0°C at low pressure or at 20°C at 488bar. Higher temperature (30.0°C) with the ODS-P phase resulted in only partial separation of the diastereomers even at high pressure. Only slight resolution was observed for the mixture of diastereomers with the C30 stationary phase (Inertsil C30) at 12.0°C and 441bar, although the stationary phase afforded greater resolution for ß- and γ-tocopherol than ODS-P. A monomeric C18 stationary phase did not show any separation at 12.0°C and 463bar. The results suggest that the binding site of the polymeric ODS-P phase is selective for flexible alkyl chains that provided the longest retention for the natural form, (R,R,R) form, and the enantiomer, (S,S,S) form, of dl-α-tocopherol.

Anticancer and antiangiogenic activity of surfactant-free nanoparticles based on self-assembled polymeric derivatives of vitamin E: structure-activity relationship.

α-Tocopheryl succinate (α-TOS) is a well-known mitochondrially targeted anticancer compound, however, it is highly hydrophobic and toxic. In order to improve its activity and reduce its toxicity, new surfactant-free biologically active nanoparticles (NP) were synthesized. A methacrylic derivative of α-TOS (MTOS) was prepared and incorporated in amphiphilic pseudoblock copolymers when copolymerized with N-vinylpyrrolidone (VP) by free radical polymerization (poly(VP-co-MTOS)). The selected poly(VP-co-MTOS) copolymers formed surfactant-free NP by nanoprecipitation with sizes between 96 and 220 nm and narrow size distribution, and the in vitro biological activity was tested. In order to understand the structure-activity relationship three other methacrylic monomers were synthesized and characterized: MVE did not have the succinate group, SPHY did not have the chromanol ring, and MPHY did not have both the succinate group and the chromanol ring. The corresponding families of copolymers (poly(VP-co-MVE), poly(VP-co-SPHY), and poly(VP-co-MPHY)) were synthesized and characterized, and their biological activity was compared to poly(VP-co-MTOS). Both poly(VP-co-MTOS) and poly(VP-co-MVE) presented triple action: reduced cell viability of cancer cells with little or no harm to normal cells (anticancer), reduced viability of proliferating endothelial cells with little or no harm to quiescent endothelial cells (antiangiogenic), and efficiently encapsulated hydrophobic molecules (nanocarrier). The anticancer and antiangiogenic activity of the synthesized copolymers is demonstrated as the active compound (vitamin E or α-tocopheryl succinate) do not need to be cleaved to trigger the biological action targeting ubiquinone binding sites of complex II. Poly(VP-co-SPHY) and poly(VP-co-MPHY) also formed surfactant-free NP that were also endocyted by the assayed cells; however, these NP did not selectively reduce cell viability of cancer cells. Therefore, the chromanol ring of the vitamin E analogues has an important role in the biological activity of the copolymers. Moreover, when succinate moiety is substituted and vitamin E is directly linked to the macromolecular chain through an ester bond, the biological activity is maintained.

Total synthesis of (R,R,R)-α-tocopherol through asymmetric Cu-catalyzed 1,4-addition.

By introducing a disposable activating substituent at C-3, the asymmetric 1,4-addition to a notoriously unreactive 2-substituted chromenone was achieved with high levels of (2R)-stereoselectivity in the presence of a chiral Cu(I)-phosphoramidite complex as a catalyst. This paved the way for an efficient and conceptually novel synthesis of (R,R,R)-α-tocopherol from readily available starting materials.

Besting vitamin E: sidechain substitution is key to the reactivity of naphthyridinol antioxidants in lipid bilayers.

A series of naphthyridinol analogs of α-tocopherol (α-TOH, right) with varying sidechain substitution was synthesized to determine how systematic changes in the lipophilicity of these potent antioxidants impact their radical-trapping activities in lipid bilayers, regenerability by water-soluble reductants, and binding to human tocopherol transport protein (TTP). The activities of the naphthyridinols were assayed in phosphatidylcholine unilamellar liposomes using a recently developed high-throughput assay that employs a boron dipyrromethene conjugate of α-TOH (H(2)B-PMHC) that undergoes fluorescence enhancement upon oxidation. The naphthyridinols afforded a dose-dependent protection of H(2)B-PMHC consistent with unprecedented peroxyl radical-trapping activity in lipid bilayers. While sidechain length and/or branching had no effect on their apparent reactivity, it dramatically impacted reaction stoichiometry, with more lipophilic compounds trapping two peroxyl radicals and more hydrophilic compounds trapping significantly less than one. It is suggested that the less lipophilic compounds autoxidize rapidly in the aqueous phase and that preferential partitioning of the more lipophilic compounds to the bilayer protects them from autoxidation. The cooperativity of a lipophilic naphthyridinol with water-soluble reducing agents was also studied in liposomes using H(2)B-PMHC and revealed superior regenerability by each of ascorbate, N-acetylcysteine, and urate when compared to α-TOH. Binding assays with human TTP, a key determinant of the bioavailability of the tocopherols, reveal that the naphthyiridinols can be very good ligands for the protein. In fact, naphthyridinols with sidechains of eight or more carbons had affinities for TTP which were similar to, and in one case 10-fold better than, α-TOH.

New synthetic route to N-tocopherol derivatives: synthesis of pyrrolopyridinol analogue of α-tocopherol from pyridoxine.

A new synthetic route to pyrrolopyridinol antioxidants from easily accessible pyridoxine was developed which includes phase-transfer catalytic alkylation and intramolecular Cu(I)-catalyzed amination as key steps.

Asymmetric synthesis and biological activity of nor-α-tocopherol, a new vitamin E analogue.

The vitamin E analogues (2R,4'R,8'R)-nor-α-tocopherol (94 % de) and (2RS,4'R,8'R)-nor-α-tocopherol have been synthesized from (all R)-hexahydrofarnesol and phytol, respectively. According to in vitro experiments with murine macrophages nor-α-tocopherol is an anti-inflammatory compound more potent than α-tocopherol.

Recent biomimetic and organocatalytic syntheses of alpha-tocopherol.

We report here on our efforts to develop new strategies for the synthesis of alpha-tocopherol, the biologically most significant member of the vitamin E family. This review comprises five new methods to generate the chiral chromane of alpha-tocopherol with overall up to 29% yield from commercially available material and up to 94% de.

Design, synthesis, and evaluation of an α-tocopherol analogue as a mitochondrial antioxidant.

An efficient synthesis has provided access to a novel α-tocopherol analogue (2), as well as its trifluoroacetate salt and acetate ester. An annulation reaction was used to establish the pyridinol core structure and a Stille coupling reaction was employed for conjugation with the tocopherol side chain. This analogue was shown to suppress the levels of reactive oxygen species in cultured cells, and to quench peroxidation of mitochondrial membranes.