Mucin-mediated nanocarrier disassembly for triggered uptake of oligonucleotides as a delivery strategy for the potential treatment of mucosal tumours.

This work demonstrates gastric mucin-triggered nanocarrier disassembly for release of antisense oligonucleotides and consequent unassisted cellular entry as a novel oral delivery strategy. A fluorescence activation-based reporter system was used to investigate the interaction and mucin-mediated disassembly of chitosan-based nanocarriers containing a 13-mer DNA oligonucleotide with a flanked locked RNA nucleic acid gapmer design. Gastric mucins were shown to trigger gapmer release from nanocarriers that was dependent on the interaction time, mucin concentration and N : P ratio with a maximal release at N : P 10. In contrast to siRNA, naked gapmers exhibited uptake into mucus producing HT-MTX mono-cultures and HT-MTX co-cultured with the carcinoma epithelial cell line Caco-2. Importantly, in vivo gapmer uptake was observed in epithelial tissue 30 min post-injection in murine intestinal loops. The findings present a mucosal design-based system tailored for local delivery of oligonucleotides that may maximize the effectiveness of gene silencing therapeutics within tumours at mucosal sites.

Surface analysis of PEGylated nano-shields on nanoparticles installed by hydrophobic anchors.

PURPOSE: This work describes a method for functionalisation of nanoparticle surfaces with hydrophilic "nano-shields" and the application of advanced surface characterisation to determine PEG amount and accumulation at the outmost 10 nm surface that is the predominant factor in determining protein and cellular interactions. METHODS: Poly(lactic-co-glycolic acid) (PLGA) nanoparticles were prepared with a hydrophilic PEGylated "nano-shield" inserted at different levels by hydrophobic anchoring using either a phospholipid-PEG conjugate or the copolymer PLGA-block-PEG by an emulsification/diffusion method. Surface and bulk analysis was performed including X-ray photoelectron spectroscopy (XPS), nuclear magnetic resonance spectroscopy (NMR) and zeta potential. Cellular uptake was investigated in RAW 264.7 macrophages by flow cytometry. RESULTS: Sub-micron nanoparticles were formed and the combination of (NMR) and XPS revealed increasing PEG levels at the particle surface at higher PLGA-b-PEG copolymer levels. Reduced cellular interaction with RAW 264.7 cells was demonstrated that correlated with greater surface presentation of PEG. CONCLUSION: This work demonstrates a versatile procedure for decorating nanoparticle surfaces with hydrophilic "nano-shields". XPS in combination with NMR enabled precise determination of PEG at the outmost surface to predict and optimize the biological performance of nanoparticle-based drug delivery.

Investigation of particle-functionalized tissue engineering scaffolds using X-ray tomographic microscopy.

A low-density, porous chitosan/poly-(dl-lactide-co-glycolide) (PLGA) microparticle composite scaffold was produced by thermally induced phase separation followed by lyophilization, to provide a bicontinuous microstructure potentially suitable for tissue engineering and locally controlled drug release. PLGA particles were mixed into the chitosan solution and subsequent phase separation during chitosan solidification forced PLGA particles into chitosan phase (Plateau borders). The distributions of volume, surface area, and elongation of 15,422 inclusions of agglomerated PLGA particles were calculated and approximated with log-normal distribution functions from nanotomography reconstructions. Cluster analysis revealed a homogenous inclusion distribution throughout the scaffold. The spatial location and orientation of individual inclusions within the Plateau borders of the scaffold were determined and from these the nearest-neighbor inter-inclusion distance distribution calculated, showing a mean of 2.5 microm. The depth of the inclusions in Plateau borders peaks at 700 or 125 nm, respectively, indicating a step-wise drug release from inclusions successively exposed during scaffold decomposition. Particle diameter ranged from 400 nm to 3 microm and inclusion Feret lengths ranged from 800 nm to 12 microm. These findings on composite morphology and distribution of inclusions are fundamental for predicting scaffold deterioration and particle-mediated drug release during ex vivo and in vivo cell cultivation.

How much did cold shock and swimming failure contribute to drowning deaths in the fishing industry in British Columbia 1976-2002?

BACKGROUND: The Workers' Compensation Board of British Columbia requested a retrospective analysis of all fishermen's deaths from immersion in water in British Columbia. AIMS: To identify the underlying cause of drowning and make recommendations to improve safety in the fishing industry. METHOD: Eighty-nine inshore and offshore fishing accidents were analysed. Where possible, deaths were classified into the four stages of cold-water immersion: cold shock, swimming failure, hypothermia and post-rescue collapse. Other factors that led up to the drowning were also identified. RESULTS: One hundred and thirty fishermen died from immersion between 1976 and 2002. One hundred and twenty-eight drownings were certified by the coroner as drowning or drowning/hypothermia and two were certified as cardiac event after immersion. The underlying causes of drownings were reclassified as: cold shock (5.4%), swimming failure (5.4%), hypothermia (5.4%), post-rescue collapse (0.8%), cardiac event (0.8%) and drowning/other (10%). In the remaining 72.2% of deaths, there was insufficient information to determine an underlying cause. All deaths occurred in water below 17.5 degrees C but 95% were in water less than 15 degrees C. CONCLUSIONS: Immersion in water below 15 degrees C is dangerous and this should be emphasized on marine survival courses. Accident investigators, coroners and pathologists need a common checklist to record vital data. A recommended format is included as Supplementary data available at Occupational Medicine Online. Fishermen should be educated about the dangers of sudden, unexpected immersion in cold water. Consideration should be given to making marine survival courses mandatory for fishermen.

Determination of the selenium requirement in kittens.

The purpose of this study was to determine the selenium (Se) requirement in kittens. Thirty-six specific-pathogen-free kittens (9.8 weeks old) were utilized in a randomized complete block design to determine the Se requirement in cats with gender and weight used as blocking criteria. Kittens were fed a low Se (0.02 mg/kg Se) torula yeast-based diet for 5 weeks (pre-test) after which an amino acid-based diet (0.027 mg Se/kg diet) was fed for 8 weeks (experimental period). Six levels of Se (0, 0.05, 0.075, 0.10, 0.20 and 0.30 mg Se/kg diet) as Na2SeO3 were added to the diet and were used to construct a response curve. Response variables included Se concentrations and Se-dependent glutathione peroxidase activities (GSHpx) in plasma and red blood cells (RBC) as well as plasma total T3 (TT3) and total T4 (TT4). No significant changes in food intake, weight gain or clinical signs of Se deficiency were noted. Estimates of the kitten's Se requirement (i.e. breakpoints) were determined for RBC and plasma GSHpx (0.12 and 0.15 mg Se/kg diet, respectively), but no definitive breakpoint was determined for plasma Se. Plasma TT3 increased linearly, whereas plasma TT4 and the ratio of TT4 : TT3 decreased in a quadratic fashion to dietary Se concentration. The requirement estimate determined in this study (0.15 mg Se/kg) for kittens is in close agreement with other species. As pet foods for cats contain a high proportion of animal protein with a Se bioavailability of 30%, it is recommended that commercial diets for cats contain 0.5 mg Se/kg DM.

A low-selenium diet increases thyroxine and decreases 3,5,3'triiodothyronine in the plasma of kittens.

The effect of a low-selenium diet on thyroid hormone metabolism was investigated in growing kittens. Twelve specific-pathogen-free kittens with ages ranging from 16 to 18 weeks were divided into two groups of equal number with equal sex distribution in each group. One group was fed a yeast-based low-selenium diet (0.02 mg Se/kg diet) while the other group was fed the same diet supplemented with Na2SeO3 at 0.4 mg Se/kg diet for 8 weeks. Food intake, body weight and body weight gain were not affected by the low-Se diet during the study period. However, kittens given the low-Se diet had significantly reduced plasma selenium concentration and glutathione peroxidase activity. Plasma total thyroxine (T4) increased and total 3,5,3'triiodothyronine (T3) decreased significantly in kittens fed the low-Se diet at the end of the study. These results suggest that type I deiodinase in cats is a selenoprotein- or a selenium-dependent enzyme.

What factors are associated with local enforcement of laws banning illegal tobacco sales to minors? A study of 182 law enforcement agencies in California.

OBJECTIVES: The aims of this study were to understand the attitudes of local law enforcement staff regarding policies to reduce youth access to tobacco, to determine what proportion of these agencies have conducted unannounced compliance checks in the past year, and to examine factors associated with conducting compliance checks. METHODS: A written questionnaire was completed by 182 law enforcement officials representing 200 cities and counties in California (some officials represented multiple jurisdictions). Logistic regression analyses were conducted to identify factors associated with conducting compliance checks. RESULTS: Only 36% of local enforcement agencies reported conducting one or more compliance checks in the previous year. Agencies were more likely to conduct compliance checks if they perceived fewer barriers to enforcement, reported more frequent collaboration with other community agencies to enforce youth access laws, and believed that youth access to tobacco is a problem in their community. CONCLUSIONS: Because active enforcement of youth access laws using unannounced compliance checks has been shown to reduce the rate of illegal tobacco sales to minors and may reduce youth smoking, efforts to increase the level of enforcement should be promoted. These study results may be used to inform efforts to encourage local agencies to enforce existing youth access laws.

Ethnic variation in peer influences on adolescent smoking.

Previous research has indicated that the influence of peers on adolescent smoking may differ across ethnic groups. Although many studies have focused on African Americans, Hispanics, and Whites, few studies have included Asian Americans, Pacific Islanders, and multi-ethnic adolescents as distinct groups. Using data from a statewide sample of 5870 eighth-grade adolescents in California, this study examined ethnic differences in the association between peer influence variables and smoking behavior and susceptibility. Informational peer influence (best friends' smoking behavior) and normative peer influence (prevalence estimates of peer smoking) were investigated. We hypothesized that informational peer influences would be stronger among Whites (whose families originate primarily from the individualistic cultures of the USA and Western Europe) than among Asian Americans, Pacific Islanders, Hispanics, and African Americans (whose families originate primarily from collectivist cultures). Conversely, we hypothesized that normative peer influences would be stronger among ethnic minority adolescents from collectivist cultural backgrounds than among Whites. Consistent with previous studies, friends' smoking and prevalence estimates of peer smoking were risk factors for past 30-day smoking and susceptibility to smoking across ethnic groups. The influence of friends' smoking behavior was stronger among Whites than among several other groups: Pacific Islanders, African Americans, and Hispanic/Latinos. The influence of prevalence estimates of peer smoking was stronger among Whites than among multiethnic adolescents. Results indicate that cultural factors may play a role in peer influences on smoking initiation. Smoking prevention interventions for adolescents should address the differences in peer influences across ethnic groups.

Influence of hydrophilicity of cationic polymers on the biophysical properties of polyelectrolyte complexes formed by self-assembly with DNA.

To investigate the possibility of producing charge-neutral gene delivery complexes with extended, non-particulate structures, DNA was allowed to self-assemble with a series of hydrophilic cationic polymers containing quaternary charged trimethylammonio ethylmethacrylate (TMAEM, 5, 15, 50, 100 mol%) copolymerised with hydrophilic N-(2-hydroxypropyl)methacrylamide (HPMA, 95, 85, 50, 0 mol%, respectively). Copolymers were all able to bind DNA, assessed using ethidium bromide fluorescence, although copolymers with low TMAEM content did not expel ethidium bromide. Increasing TMAEM content of the copolymers changed the morphology of the complexes from extended (5-15 mol% TMAEM), through partially condensed particles (50 mol%) to discrete nanoparticles (100 mol% TMAEM). Complexes based on copolymers with low TMAEM content (5-50 mol%) showed less resistance to degradation by nucleases and lower surface charge (21.2+/-5.9-45.1+/-3.9 mV) than those formed using 100 mol% TMAEM (57.8+/-8.2 mV). They also showed significantly less association with phagocytic cells in vitro (human leucocytes, uptake decreased by up to 92.3%; murine peritoneal macrophages, uptake decreased by up to 69.6%), although in vivo their hepatic accumulation was only slightly decreased (maximum decrease 27.6%). Finding the appropriate balance of hydrophilicity and stability is key to development of effective vectors for gene delivery.

Opinion leaders' support for tobacco control policies and participation in tobacco control activities.

OBJECTIVES: Because opinion leaders hold positions from which they may influence tobacco control efforts, this study examined their support for tobacco control policies and their involvement in tobacco control activities. METHODS: A telephone survey was administered to 712 California opinion leaders who were randomly selected from constructed lists representing 8 types of organizations: health, education, law enforcement, media, government, business, ethnic, and youth. Hierarchical regression analysis was used to identify predictors of support for and participation in tobacco control activities. RESULTS: Approximately one half to two thirds of opinion leaders supported the tobacco control policies queried; 60% reported involvement in tobacco control-related activities during the previous year. Organizational affiliation was a strong predictor of support and involvement, with leaders from health and educational organizations reporting the highest levels and business and media leaders reporting the lowest. Tobacco issue involvement variables (e.g., having a friend or family member with a smoking-related illness) were significantly associated with the outcomes, while sociodemographics, for the most part, were not. CONCLUSIONS: Study results can be used to mobilize opinion leaders' support for tobacco control more effectively.

Steric stabilization of poly-L-Lysine/DNA complexes by the covalent attachment of semitelechelic poly[N-(2-hydroxypropyl)methacrylamide].

The concept of steric stabilization was utilized for self-assembling polyelectrolyte poly-L-lysine/DNA (pLL/DNA) complexes using covalent attachment of semitelechelic poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA). We have examined the effect of coating of the complexes with pHPMA on their physicochemical stability, phagocytic uptake in vitro, and biodistribution in vivo. The coated complexes showed stability against aggregation in 0.15 M NaCl and reduced binding of albumin, chosen as a model for the study of the interactions of the complexes with plasma proteins. The presence of coating pHPMA had no effect on the morphology of the complexes as shown by transmission electron microscopy. However, results of the study of polyelectrolyte exchange reactions with heparin and pLL suggested decreased stability of the coated complexes in these types of reactions compared to uncoated pLL/DNA complexes. Coated complexes showed decreased phagocytic capture by mouse peritoneal macrophages in vitro. Decreased phagocytosis in vitro, however, did not correlate with results of in vivo study in mice showing no reduction in the liver uptake and no increase in the circulation times in the blood. We propose that the rapid plasma elimination of coated pLL/DNA complexes is a result of binding serum proteins and also of their low stability toward polyelectrolyte exchange reactions as a consequence of their equilibrium nature.

Physicochemical and biological characterisation of an antisense oligonucleotide targeted against the bcl-2 mRNA complexed with cationic-hydrophilic copolymers.

The aim of this study was to evaluate the use of cationic-hydrophilic copolymers for self-assembly with antisense oligonucleotides targeted to the bcl-2 mRNA in order to improve their biocompatibility and modulation of their pharmacokinetics for greater therapeutic usefulness. Examination of the ability of poly(trimethylammonioethyl methacrylate chloride)-poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA-b-pTMAEM) block copolymers to condense the oligonucleotide by fluorescence and electrophoresis techniques showed that complexes were formed more efficiently than with copolymers containing poly(ethylene glycol) blocks grafted onto the backbone of poly(L-lysine) (pLL-g-pEG). In addition, the copolymer pTMAEM-b-pHPMA produced oligonucleotide complexes with the most favourable physicochemical properties appropriate for in vivo applications. The complexes were small (approximately 36 nm in diameter), with low surface charge as measured by zeta potential, relatively stable to physiological salt conditions and could be formed at a DNA concentration of 500 microg/ml. Complex formation with the copolymer pTMAEM-b-pHPMA or pLL-g-pEG reduced the urinary clearance of the oligonucleotide after intravenous injection into mice. However after 30 min, the oligonucleotide complexes were cleared from the bloodstream. These results indicate that for the systemic delivery of oligonucleotides the polymer-derived complexes are not stable enough for prolonged circulation. Instead, these complexes may be more suitable for localised in vivo applications.

Decreased binding to proteins and cells of polymeric gene delivery vectors surface modified with a multivalent hydrophilic polymer and retargeting through attachment of transferrin.

Binding of serum proteins to polyelectrolyte gene delivery complexes is thought to be an important factor limiting bloodstream circulation and restricting access to target tissues. Protein binding can also inhibit transfection activity in vitro. In this study a multivalent reactive hydrophilic polymer has been used to inhibit protein binding. This polymer is based on poly-[N-(2-hydroxypropyl)methacrylamide] (pHPMA) bearing pendent oligopeptide (Gly-Phe-Leu-Gly) side chains terminated in reactive 4-nitrophenoxy groups (8.6 mol%). The polymer reacts with the primary amino groups of poly(L-lysine) (pLL) and produces a hydrophilic coating on the surface of pLL.DNA complexes (as measured by fluorescamine). The resulting pHPMA-coated complexes show a decreased surface charge (from +14 mV for pLL.DNA complexes to -25 mV for pHPMA-modified complexes) as measured by zeta potential analysis. The pHPMA-coated complexes also show a slightly increased average diameter (approximately 90 nm compared with 60 nm for pLL. DNA complexes) as viewed by atomic force and transmission electron microscopy and around 100 nm as viewed by photon correlation spectroscopy. They are completely resistant to protein interaction, as determined by turbidometry and SDS-polyacrylamide gel electrophoresis analysis of complexes isolated from plasma, and show significantly decreased nonspecific uptake into cells in vitro. Spare reactive ester groups can be used to conjugate targeting ligands (e.g. transferrin) on to the surface of the complex to provide a means of tissue-specific targeting and transfection. The properties of these complexes therefore make them promising candidates for targeted gene delivery, both in vitro and potentially in vivo.

The relationship between home smoking bans and exposure to state tobacco control efforts and smoking behaviors.

PURPOSE: To examine the relationship between home smoking bans and adult smokers' exposure to the statewide California Tobacco Control Program (TCP) and their cigarette smoking behavior. DESIGN: Cross-sectional survey that was part of the statewide Independent Evaluation of the California Tobacco Control, Prevention and Education Program. SETTING: Random telephone interviews within 18 California counties. SUBJECTS: A representative sample of 1315 adult smokers, aged 25 years and older. MEASURES: The telephone survey included questions about smoking behavior, quitting smoking, exposure to tobacco control program components, home smoking rules, and attitudes related to tobacco use and environmental tobacco smoke (ETS). RESULTS: Smokers with a home smoking ban were twice as likely (OR = 2.29; 95% CI 1.22, 4.29) to have heard of TCP community programs and three times more likely (OR = 3.18; 95% CI 1.34, 7.57) to have seen and talked about the ETS media spot than smokers with no home smoking policy. Multivariate regression models indicated that having a home smoking ban was related to smoking fewer cigarettes per day and greater interest in quitting smoking compared with smokers with no smoking rules in the home (p < .05). CONCLUSIONS: These findings suggest that smokers reporting exposure to the California TCP were more likely to have restrictive home smoking policies and that more restrictive home smoking policies were associated with reduced smoking behavior.

Attitudes toward anti-tobacco policy among California youth: associations with smoking status, psychosocial variables and advocacy actions.

To prevent smoking and exposure to environmental tobacco smoke, California has implemented anti-tobacco policies, including laws restricting youth access to tobacco, and smoking bans in workplaces, schools, restaurants and bars. Although studies have examined adults' attitudes toward anti-tobacco policies, little is known about adolescents' awareness of and support for these policies. This study examined attitudes toward anti-tobacco policies in a sample of 6887 10th grade California adolescents. Awareness of anti-tobacco policies was highest among current smokers and lowest among susceptible never-smokers. Support for anti-tobacco policies was highest among non-susceptible never-smokers and lowest among current smokers. Policy awareness and support were significantly associated with psychosocial tobacco-related variables (e.g. perceived consequences of smoking, friends' smoking, perceived access to cigarettes, prevalence estimates of smoking among peers, cigarette offers and cigarette refusal self-efficacy). Policy awareness and support were associated with the probability of performing advocacy actions against tobacco use. Although these results cannot prove a causal association, they suggest that adolescents' attitudes toward anti-tobacco policies may play a role in their decisions about smoking. Tobacco control and education programs should include information about existing anti-tobacco policies, and should educate youth about the importance and benefits of anti-tobacco policies.

Smoking bans in the home and car: Do those who really need them have them?

BACKGROUND: This paper addresses the question of whether individuals who are most in need of household and car smoking bans, such as individuals with children living at home or who have many friends who smoke, are the ones who have them. METHOD: A representative sample of 6985 California adults ages 18 and older participated in telephone interviews. RESULTS: Overall, 76% of adults report having home smoking bans and 66% have car smoking bans. Being a smoker or African American, not having children in the home, having more friends who smoke, and lower household income were associated with lower prevalence of both home and car smoking bans (P < 0.01). In multivariate analyses, nonsmokers were 7.9 (95% CI = 3.56, 17.31) times more likely to have a home smoking ban when none of their friends were smokers compared to when most of their friends were smokers. Among smokers, there was an interaction between having children at home and the proportion of friends who smoke. Only 27 to 55% of smokers had home smoking bans unless most of their friends were smokers, then the odds of having a ban were 6.1 (95% CI = 2.76, 13.68) times higher for smokers with children (67% with home bans) than for smokers without children at home (25% with home bans). CONCLUSIONS: Efforts to increase home and car smoking bans for nonsmokers who have friends who smoke and smokers with children living at home are needed.

Which adults do underaged youth ask for cigarettes?

OBJECTIVES: This study identified adults' demographic and smoking behavior characteristics that are related to being asked to provide tobacco to a minor. METHODS: Telephone interviews were conducted with 6352 California adults. Predictors included age, sex, household income, and smoking status. RESULTS: Only 10.1% of California adults had been asked to provide tobacco to a minor in the previous year. Fewer than 3% of individuals 55 years and older had been asked to provide tobacco, but among younger smokers 59.0% of 18- and 19-year-olds and 39.3% of 20- to 24-year-olds had been approached. CONCLUSIONS: Interventions to reduce the social availability of tobacco are needed.

Effect of magnesium-deficient diet on serum and urine magnesium concentrations in healthy cats.

OBJECTIVE: To evaluate the efficacy of using serum total and ionized magnesium (Mg) concentrations and urine Mg concentrations to identify Mg deficiency in cats. ANIMALS: 6 healthy castrated male cats. PROCEDURE: A Mg-replete diet was fed for 37 days, followed by a Mg-deficient diet for 37 days. On days 1, 3, and 7 of the last week of each diet, serum ionized and total Mg concentrations were determined; in addition, urine Mg concentration was determined each day of the last week. Serum total and ionized Mg concentrations were compared with urine Mg concentration, amount of Mg excreted during 24 hours (24-hour urine Mg excretion), ratio of urine Mg concentration to urine creatinine concentration (Umg:Ucr), and urinary fractional excretion of Mg (FEmg) to determine which variable best predicted Mg status. RESULTS: Cats fed Mg-deficient diets had significantly lower serum total and ionized Mg concentrations and 24-hour urine Mg excretion values, compared with cats fed Mg-replete diets. Serum total Mg concentration was the best predictor of Mg status. Twenty-four-hour urine Mg excretion was a repeatable, reliable measurement and had the best correlation with serum total Mg concentration. Serum total Mg concentration also correlated with urine Mg concentration, Umg:Ucr, and FEmg. CONCLUSIONS AND CLINICAL RELEVANCE: Serum total and ionized Mg concentrations can be used to identify cats with dietary-induced Mg deficiencies. Twenty-four-hour urine Mg excretion and urine Mg concentration correlated best with serum total Mg concentration and, therefore, may be the most useful urine variables for identifying Mg deficiency.

Beliefs about the risks of guns in the home: analysis of a national survey.

OBJECTIVES: While epidemiological evidence suggests homes with guns are more likely to be the site of a suicide or homicide than homes without guns, the public's perception of these risks remains unknown. This study assesses the prevalence of the belief that homes with guns are safer than homes without guns, and factors associated with this belief. METHODS: Telephone interviews were conducted with a random sample of 4138 registered voters in urban areas in the US. Multinomial logistic regression was used to assess correlates of beliefs about the safety of keeping a gun in the home. RESULTS: Twenty nine per cent of respondents believed keeping a gun in the home makes the home more safe, 40% said less safe, 23% said it depends, and 9% were unsure. The belief that a home is more safe with a gun was associated with being male, young, completing 12 years or fewer of education, having no children living at home, Republican party affiliation, and low levels of trust in the police for protection. Prior exposure to violence and fear of victimization were not associated with the outcome. CONCLUSIONS: Findings may increase understanding about the public's perception of the risk in keeping guns in the home and assist educational efforts to decrease the risk of these injuries.