Costas Demetzos

Nanoliposomes and Their Applications in Food Nanotechnology

Food nanotechnology involves the utilization of nanocarrier systems to stabilize the bioactive materials against a range of environmental and chemical changes as well as to improve their bioavailability. Nanoliposome technology presents exciting opportunities for food technologists in areas such as encapsulation and controlled release of food materials, as well as the enhanced bioavailability, stability, and shelf-life of sensitive ingredients. Liposomes and nanoliposomes have been used in the food industry to deliver flavors and nutrients and, more recently, have been investigated for their ability to incorporate antimicrobials that could aid in the protection of food products against microbial contamination. In this paper, the main physicochemical properties of liposomes and nanoliposomes are described and some of the industrially applicable methods for their manufacture are reviewed. A summary of the application of nanoliposomes as carrier vehicles of nutrients, nutraceuticals, enzymes, food additives, and food antimicrobials is also presented.

Essential oil analysis and antimicrobial activity of eight Stachys species from Greece.

The volatile composition of eight Stachys species has been studied. The investigated taxa were St. alopecuros (L.) Bentham., St. scardica (Griseb.) Hayek, St. cretica L. ssp. cretica, St. germanica L. ssp. heldreichii (Boiss.) Hayek, St. recta L., St. spinulosa L., St. euboica Rech. and St. menthifolia Vis., growing wild in Greece. The essential oils were obtained by hydrodistillation in a modified Clevenger-type apparatus, and their analyses were performed by GC and GC-MS. Identification of the substances was made by comparison of mass spectra and retention indices with literature records. Sesquiterpene hydrocarbons were shown to be the main group of constituents of all taxa. Furthermore, the obtained essential oils were tested against the following six bacteria: Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 35210), Bacillus subtilis (ATCC 10907), Bacillus cereus (clinical isolates), Micrococcus flavus (ATCC 10240), Staphylococcus epidermidis (ATCC 2228), as well as against the following five fungi: Aspergillus niger (ATCC 6275), Penicillium ochrochloron (ATCC 9112), Epidermophyton floccosum (clinical isolates), Candida albicans (clinical isolates) and Trichophyton mentagrophytes (clinical isolates). The tested essential oils showed better activity against bacterial species than against fungi. Pseudomonas aeruginosa was the most resistant strain, as none of the essential oils was active against this strain. The essential oil of St. scardica has been proven most active against both bacteria and fungi.

Differential Scanning Calorimetry (DSC): A Tool to Study the Thermal Behavior of Lipid Bilayers and Liposomal Stability

Thermodynamical techniques are applied for determining the thermal stress of medicinal compounds of the excipients as well as their interactions during the formulation process. The physicochemical properties and the stability of the medicinal products could be measured as a function of temperature or time using thermal analysis. Differential Scanning Calorimetry (DSC) is a suitable thermal analysis technique for determining the purity, the polymorphic forms and the melting point of a sample in the Pharmaceutical Industry. It is also considered as a tool to study the thermal behavior of lipid bilayers and of lipidic drug delivery systems, like liposomes by measuring thermodynamic parameters (i.e. ΔH and Tm), which affect the stability of the liposomal suspension under given storage conditions.

The effect of sclareol on growth and cell cycle progression of human leukemic cell lines.

Sclareol, a labdane-type diterpene, was tested for cytotoxic effect against a panel of established human leukemic cell lines. The compound showed an IC50 lower than 20 microg/ml in most cell lines tested, while it was higher for resting peripheral blood mononuclear leukocytes (PBML). Furthermore, the compound was tested for cytostatic activity against four of the leukemic cell lines used. At a concentration of 20 microg/ml the compound showed a significant cytostatic effect as soon as 4 h after continuous incubation against two from B and two from T lineage cell lines. The morphology and the kind of death induced from sclareol in three cell lines, was also investigated. The effect of sclareol on the cell cycle progression of two cell lines, using flow cytometry, was examined. The results show that sclareol kills cell lines, through the process of apoptosis. The appearance of the apoptotic signs is time and dose dependent. From the flow cytometry experiments, a delay of the cell population on G0/1 seems to take place. This is the first report, that a labdane type diterpene kills tumor cells via a phase specific mechanism which induces apoptosis.

Self‐preserving cosmetics

Preservatives are added to products for two reasons: first, to prevent microbial spoilage and therefore to prolong the shelf life of the product; second, to protect the consumer from a potential infection. Although chemical preservatives prevent microbial growth, their safety is questioned by a growing segment of consumers. Therefore, there is a considerable interest in the development of preservative‐free or self‐preserving cosmetics. In these formulations traditional/chemical preservatives have been replaced by other cosmetic ingredients with antimicrobial properties that are not legislated as preservatives according to the Annex VI of the Commission Directive 76/768/EEC and the amending directives (2003/15/EC, 2007/17/EC and 2007/22/EC). ‘Hurdle Technology’, a technology that has been used for the control of product safety in the food industry since 1970s, has also been applied for the production of self‐preserving cosmetics. ‘Hurdle Technology’ is a term used to describe the intelligent combination of different preservation factors or hurdles to deteriorate the growth of microorganisms. Adherence to current good manufacturing practice, appropriate packaging, careful choice of the form of the emulsion, low water activity and low or high pH values are significant variables for the control of microbial growth in cosmetic formulations. This paper describes the application of the basic principles of ‘Hurdle Technology’ in the production of self‐preserving cosmetics. Multifunctional antimicrobial ingredients and plant‐derived essential oils and extracts that are used as alternative or natural preservatives and are not listed in Annex VI of the Cosmetic Directive are also reported.

Labdane-type diterpenes: Chemistry and biological activity

Abstract The terpenoids are a class of natural products with biological activity. The number of isoprene units from which they are biogenetically composed is used for their classification. The diterpenes are another group from which several compounds with high biological activity have been produced. The most studied plant families are Asteraceae and Labiatae, as well as Conifers, which are the main sources of diterpenes. The lack of, or insufficient, chromatographic data (RI, Rt) as well as of reference compounds for most labdanes is the main reason for their absence from the chemical fingerprints of plantextracts. Also their use as chemotaxonomic markers within the species in which they occur is limited. The configuration at the C-13 carbon atom has been investigated, especially in the case of manoyl oxide, as well as the various isomers, the ratio of which determines antimicrobial activity. A variety of biological activities have been encountered in labdane diterpenes such as antibacterial, antifungal, antiprotozoal, enzyme inducing, anti-inflammatory activities and modulation of immune cell functions. More recent studies have shown that labdane exhibits significant cytotoxic and cytostatic effects against leukemic cell lines of human origin and interferes with the biochemical pathways of apoptosis and the cell cycle phases, as well as with the expression of several protooncogenes such as c-myc and bcl-2 . This report underlines the role of these compounds, not only as toolsfor the study of the biochemistry and regulation of biochemical and metabolic pathways of mammalian cellular systems, but also as potential pharmacological agents in the fight against diseases such as cancer and heart disorders.

Labdane type diterpenes down-regulate the expression of c-Myc protein, but not of Bcl-2, in human leukemia T-cells undergoing apoptosis.

Sclareol (1) and ent-3beta-hydroxy-13-epi-manoyl oxide (2) belong to the labdane type diterpenes. They were isolated from the leaves and from the fruits of Cistus creticus subsp. creticus, and were found to be active against human leukemic cell lines. Compound 2 was converted to its thiomidazolide derivative (3). Compounds 1 and 3 were found to induce apoptotic cell death in human T-cell leukemia lines and to interfere with their cell cycle, arresting cells at G(0/1) phase. Apoptosis can involve the activation and/or suppression of critical genes such as c-myc whose reduction or its inappropriate expression can be associated with induction of cell death and bcl-2 whose activation prevents apoptosis in the latter case. In order to detect any concomitant effect (1 and 3) upon c-myc and bcl-2 oncogene expression, we performed Western blot analysis to determine the levels of expression of these two genes upon treatment with the above compounds. Western blot analysis showed that of c-myc proto-oncogene levels were markedly reduced before massive apoptosis ensued in H33AJ-JA1 and MOLT3 cells, while bcl-2 expression remained unaffected. Thus, induction of apoptosis due to compounds 1 and 3 in these T-cell leukemic cell lines is preceded by c-myc down regulation and furthermore sustained bcl-2 expression does not rescue cells from apoptosis under the conditions used.

Mitochondria-targeted liposomes improve the apoptotic and cytotoxic action of sclareol

Current efforts toward improving the effectiveness of drug therapy are increasingly relying on drug-targeting strategies to effectively deliver bioactive molecules to their molecular targets. Pharmaceutical nanocarriers represent a major tool toward this aim, and our efforts have been directed toward achieving nanocarrier-mediated subcellular delivery of drug molecules with mitochondria as the primary subcellular target. Meeting the need for specific subcellular delivery is essential to realizing the full potential of many poorly soluble anticancer drugs. In this article, we report that mitochondria-targeted liposomes significantly improve the apoptotic and cytotoxic action of sclareol, a poorly soluble potential anticancer drug. The results support the broad applicability of our nanocarrier-mediated subcellular targeting approach as a means to improve the effectiveness of certain anticancer therapeutics.

DPPC:MPOx chimeric advanced Drug Delivery nano Systems (chi-aDDnSs): Physicochemical and structural characterization, stability and drug release studies

Chimeric advanced Drug Delivery nano Systems (chi-aDDnSs) could be defined as mixed nanosystems composed of different biomaterials that can be organized into new nanostructures that can offer advantages as drug carriers. In this work, we report on the self assembly behavior and on stability studies of chi-aDDnSs consisting of DPPC (dipalmitoylphosphatidylcholine) and poly(2-methyl-2-oxazoline)-grad-poly(2-phenyl-2-oxazoline) (MPOx) gradient copolymer in Phosphate Buffer Saline (PBS). Light scattering techniques were used in order to extract information on their physicochemical and structural characteristics (i.e. ζ-potential, Polydispersity Index (PD.I.), size/shape and morphology), while their stability was also studied as a function of gradient block copolymer content, as well as temperature. The colloidal stability of the chimeric nanovectors and their thermoresponsive behavior indicates that these nanosystems could be considered as sterically stabilized nanocontainers. DPPC:MPOx chimeric advanced Drug Delivery nano Systems were found to be effective nanocontainers for the incorporation of indomethacin (IND). The combination of gradient block copolymers with phospholipids for the development of novel chimeric nanovectors is reported for the first time and appears very promising, mostly due to the fact that the MPOx acts as a modulator for the release rate of the IND.

Sclareol induces apoptosis in human HCT116 colon cancer cells in vitro and suppression of HCT116 tumor growth in immunodeficient mice.

Labd-14-ene-8, 13-diol (sclareol) is a labdane-type diterpene, which has demonstrated significant cytotoxic activity against human leukemic cell lines, but its effect on solid tumor-derived cells is uknown. Here, we demonstrate that addition of sclareol to cultures of human colon cancer HCT116 cells results in inhibition of DNA synthesis, arrest of cells at the G1 phase of the cell cycle, activation of caspases-8, -9, PARP degradation, and DNA fragmentation, events characteristic of induction of apoptosis. Intraperitoneal (ip) administration of sclareol alone, at the maximum tolerated dose, was unable to induce suppression of growth of HCT116 tumors established as xenografts in immunodeficient SCID mice. In contrast, ip administration of liposome-encapsulated sclareol, following a specific schedule, induced suppression of tumor growth by arresting tumor cell proliferation as assessed by detecting the presence of the cell proliferation-associated nuclear protein, Ki67, in thin tumor sections. These findings suggest that sclareol incorporated into liposomes may possess chemotherapeutic potential for the treatment of colorectal and other types of human cancer.