Liposomal Tumor Targeting in Drug Delivery Utilizing MMP-2- and MMP-9-Binding Ligands.

Nanotechnology offers an alternative to conventional treatment options by enabling different drug delivery and controlled-release delivery strategies. Liposomes being especially biodegradable and in most cases essentially nontoxic offer a versatile platform for several different delivery approaches that can potentially enhance the delivery and targeting of therapies to tumors. Liposomes penetrate tumors spontaneously as a result of fenestrated blood vessels within tumors, leading to known enhanced permeability and subsequent drug retention effects. In addition, liposomes can be used to carry radioactive moieties, such as radiotracers, which can be bound at multiple locations within liposomes, making them attractive carriers for molecular imaging applications. Phage display is a technique that can deliver various high-affinity and selectivity peptides to different targets. In this study, gelatinase-binding peptides, found by phage display, were attached to liposomes by covalent peptide-PEG-PE anchor creating a targeted drug delivery vehicle. Gelatinases as extracellular targets for tumor targeting offer a viable alternative for tumor targeting. Our findings show that targeted drug delivery is more efficient than non-targeted drug delivery.

Occupational exposure in dentistry and miscarriage.

BACKGROUND: Information on the reproductive effects of chemical exposures in dental work is sparse or inconsistent. AIM: To investigate whether dental workers exposed to acrylate compounds, mercury amalgam, solvents or disinfectants are at an increased risk of miscarriage. METHODS: The study was conducted among women dental workers and a comparison group of workers occupationally unexposed to dental restorative materials. Information on pregnancies was obtained from national registers and outpatient units of hospitals. Data on occupational exposure were obtained using postal questionnaires. The final study population included 222 cases of miscarriage and 498 controls (births). An occupational hygienist assessed exposure to acrylate compounds, disinfectants and solvents. Exposure to other agents was assessed on the basis of the questionnaire data. Odds ratios (ORs) and confidence intervals (CIs) were estimated using conditional logistic regression. RESULTS: The ORs adjusted for confounding factors were increased for moderate-exposure and high-exposure categories of mercury amalgam (OR 2.0, 95% CI 1.0 to 4.1 and OR 1.3, 95% CI 0.6 to 2.5, respectively). The risk was slightly increased for the highest-exposure category of 2-hydroxyethylmethacrylate (OR 1.4, 95% CI 0.7 to 2.6) and polymethylmethacrylate dust (OR 1.4, 95% CI 0.8 to 2.4). A slightly increased risk was also detected for likely exposure to organic solvents (OR 1.4, 95% CI 0.8 to 2.3) and disinfectants (OR 1.5, 95% CI 0.9 to 2.7). CONCLUSIONS: No strong association or consistent dose-response relationship was observed between exposure to chemical agents in dental work and the risk of miscarriage. A slightly increased risk was found for exposure to mercury amalgam, some acrylate compounds, solvents and disinfectants. These findings indicate that the possibility of a weak association between exposure to these agents and an increased risk of miscarriage cannot be excluded.

Multistate worklife expectancies.

This communication reviews the demographic concept of worklife expectancy and draws the epidemiologists' attention to its usefulness in occupational health research and pension policy making. The distinctions between different analytic approaches to the quantification of expected workforce status and mobility are pointed out. A recently developed multivariate large-sample regression method for the analysis of worklife tables is placed into the general context of life tables. Given aggregated data from multiple cross-sectional or longitudinal population surveys, a multistate regression model can be used to estimate consistently marginal probabilities that a person is in a given work-health state or transition probabilities between the states and, thereby, worklife expectancies. The methodology is illustrated through the application to data from Finnish population statistics on employment, disability, retirement, and mortality. The paper closes with a discussion of the methodological issues and empirical findings on pension policy in Finland.

Binding of endostatin to phosphatidylserine-containing membranes and formation of amyloid-like fibers.

Endostatin, the 20-kDa C-terminal NC1 domain of collagen XVIII, is an endogenous inhibitor of tumor angiogenesis and tumor growth. A major problem in reconciling the many reported in vitro effects of endostatin is the lack of a high-affinity receptor, and a search for the latter continues. In accordance with the above, the molecular mechanisms of action of endostatin remain elusive. We show here that endostatin binds to membranes containing acidic phospholipids, phosphatidylserine (PS) or phosphatidylglycerol (PG). More specifically, a red shift in the fluorescence emission of Trp of endostatin in the presence of liposomes containing these anionic lipids was evident, revealing the average environment of Trps to become less hydrophobic. This shift was not observed for phosphatidylcholine (PC) liposomes, demonstrating the acidic lipid to be required. Quenching by endostatin of the fluorescence of a pyrene-labeled phospholipid analogue in PS containing membranes was seen, while there was no effect for PC liposomes. Resonance energy transfer from the Trp residues of endostatin to a dansyl-labeled phospholipid further confirmed the association of endostatin with PS-containing membranes, whereas there was no binding to PC liposomes. Intriguingly, the association of endostatin with PS-containing liposomes triggered the formation of fibers, with Congo red staining producing green birefringence characteristic for amyloid. Lipid was incorporated into these fibers, as shown by staining when a trace amount (X = 0.02) of fluorescent phospholipid analogues was present in the liposomes. No fiber formation was seen when endostatin was added to liposomes composed of PC only. Because PS has been reported to be exposed in the outer surface of the plasma membrane of cancer cells and vascular endothelial cells, our results suggest that this lipid could represent a target for endostatin in the cancer cell surface and tumors, thus suggesting a novel mechanism of its action. More specifically, analogous to a number of other cytotoxic proteins interacting with negatively charged lipids, PS-triggered fiber formation by endostatin on the surface of cancer cells would impair the permeability barrier function of the plasma membrane, resulting in cell death.

Methylation of cytochrome P4501A1 promoter in the lung is associated with tobacco smoking.

Cytochrome P4501A1 (CYP1A1), which is involved in the metabolic activation of polycyclic aromatic hydrocarbon procarcinogens derived from tobacco smoke, is induced in the lung up to 100-fold because of tobacco smoking. Our aim was to study whether promoter methylation has any role in the smoking-associated expression of CYP1A1 in human lung. Methylation of CpG sites up to 1.4 kb upstream of CYP1A1 gene was studied first by sequencing. Because methylation was observed between nucleotides -1400 and -1000, a methylation-specific single-strand conformational polymorphism method was designed for the region between nucleotides -1411 and -1295 that contains five potential methylation sites, one of them at the xenobiotic responsive element core sequence. Single-strand conformational polymorphism was used on DNA from normal lung samples and peripheral WBCs of smokers and nonsmokers, and on human lung adenocarcinoma (A549) and bronchial epithelial (Beas-2B) cell lines. In lung tissue complete or partial methylation occurred in 33% of heavy smokers (>15 cigarettes/day, n = 30), 71% of light smokers (< or =15 cigarettes/day or quitted 1-7 days earlier, n = 42), and in 98% of nonsmokers (never and ex-smokers, n = 49). Methylation was found to increase in 1-7 days after quitting smoking. In active smokers the lack of methylation in the studied region of CYP1A1 promoter was associated with a slightly higher pulmonary 7-ethoxyresorufin O-deethylase activity in the regression models allowing for the daily tobacco consumption and age. No association was observed in WBC between methylation and tobacco smoking. In lung-derived cell lines the methylation remained stable regardless of induction with benzo(a)pyrene, but a higher induction was observed in Beas-2B cells, which also had less methylation than A549 cells. The association of tobacco smoking with CYP1A1 methylation in the lung suggests that promoter methylation is involved in the regulation of CYP1A1 induction in vivo.

Glutathione S-transferases and aromatic DNA adducts in smokers' bronchoalveolar macrophages.

Interindividual differences in the expression of carcinogen-metabolizing enzymes in the lung may modify the effective dose of tobacco carcinogens in this organ. We investigated the role of detoxifying glutathione S-transferases (GST) in the formation of aromatic DNA adducts in bronchoalveolar macrophages (BAM) of active smokers. The effect of GSTs on aromatic DNA adducts was studied separately and in combination with the PAH-metabolizing cytochrome P450 enzyme, CYP3A. GSTA, GSTM3, GSTP, and CYP3A protein levels were analyzed by Western blotting, GSTM1 and GSTP1 genotypes were determined by polymerase chain reaction (PCR) based methods, and numbers of aromatic DNA adducts were measured by nuclease P1 enhanced 32P-postlabeling method in BAM of 31 active smokers. No correlation was observed between GSTA or GSTP proteins or GSTM1 or GSTP1 genotypes and the level of aromatic DNA adducts. A high or medium expression level of GSTM3 was associated with a lower level of aromatic DNA adducts in the smokers who smoked less than 20 cigarettes per day, when the effect of GSTM3 was analyzed in combination with CYP3A (regression analysis; F(6,24)=6.3, P<0.001). No protection by GSTM3 was observed in heavy smokers. High CYP3A levels, on the other hand, increased the number of DNA adducts regardless of the amount of smoking.

Observation of topical catalysis by sphingomyelinase coupled to microspheres.

Sphingomyelinase, SMase (EC 3.1.4.12), was coupled onto amino-derivatized acrylate microspheres and was shown to retain its catalytic activity. The immobilized enzyme allows one to carry out topical enzymatic reaction in a controlled manner. Accordingly, these spheres were held with a micropipet and using micromanipulator brought into contact with a giant liposome membrane composed of phosphatidylcholine and sphingomyelin (SOPC/C16:0-SM, 0.75:0.25, molar ratio), representing the substrate for the immobilized enzyme. The macroscopic consequences of the enzyme reaction were visualized using fluorescence microscopy as well as differential interference contrast microscopy. The surface contact of the giant vesicle and immobilized enzyme causes membrane microdomain formation and domain clustering (capping) in the membrane and subsequent shedding of small vesicles from the membrane into the interior of the giant liposome. The method described represents a novel approach to study enzymatic reactions and allows manipulating giant vesicles as well as cultured cells in a spatially controlled manner.