Assessment of liposome disruption to quantify drug delivery in vitro

Efficient liposome disruption inside the cells is a key for success with any type of drug delivery system. The efficacy of drug delivery is currently evaluated by direct visualization of labeled liposomes internalized by cells, not addressing objectively the release and distribution of the drug. Here, we propose a novel method to easily assess liposome disruption and drug release into the cytoplasm. We propose the encapsulation of the cationic dye Hoechst 34,580 to detect an increase in blue fluorescence due to its specific binding to negatively charged DNA. For that, the dye needs to be released inside the cell and translocated to the nucleus. The present approach correlates the intensity of detected fluorescent dye with liposome disruption and consequently assesses drug delivery within the cells.Eugénia Nogueira (SFRH/BD/81269/2011), Célia F. Cruz (SFRH/BD/ 100927/2014) and Ana Loureiro (SFRH/BD/81479/2011) hold scholarships from Fundação para a Ciência e a Tecnologia (FCT). This study was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement NMP4-LA-2009-228827 NANOFOL. This study was also supported by FEDER through POFC – COMPETE and by national funds from FCT through the project PEst-UID/ BIA/4050/2013 and the strategic funding of ID/BIO/04469/2013 unit.We thank the Immuno-haemotherapy Department of Hospital de São João (Porto, Portugal) for providing buffy coats from healthy volunteers

The amount of keratins matters for stress protection of the colonic epithelium

Keratins (K) are important for epithelial stress protection as evidenced by keratin mutations predisposing to human liver diseases and possibly inflammatory bowel diseases. A role for K8 in the colon is supported by the ulcerative colitis-phenotype with epithelial hyperproliferation and abnormal ion transport in K8-knockout (K8-/-) mice. The heterozygote knockout (K8+/-) colon appears normal but displays a partial ion transport-defect. Characterizing the colonic phenotype we show that K8+/- colon expresses ~50% less keratins compared to K8 wild type (K8+/+) but de novo K7 expression is observed in the top-most cells of the K8+/- and K8-/- crypts. The K8+/- colonic crypts are significantly longer due to increased epithelial hyperproliferation, but display no defects in apoptosis or inflammation in contrast to K8-/-. When exposed to colitis using the dextran sulphate sodium-model, K8+/- mice showed higher disease sensitivity and delayed recovery compared to K8+/+ littermates. Therefore, the K8+/- mild colonic phenotype correlates with decreased keratin levels and increased sensitivity to experimental colitis, suggesting that a sufficient amount of keratin is needed for efficient stress protection in the colonic epithelia

Dicationic Alkylammonium Bromide Gemini Surfactants. Membrane Perturbation and Skin Irritation

Dicationic alkylammonium bromide gemini surfactants represent a class of amphiphiles potentially effective as skin permeation enhancers. However, only a limited number of studies has been dedicated to the evaluation of the respective cytotoxicity, and none directed to skin irritation endpoints. Supported on a cell viability study, the cytotoxicity of gemini surfactants of variable tail and spacer length was assessed. For this purpose, keratinocyte cells from human skin (NCTC 2544 cell line), frequently used as a model for skin irritation, were employed. The impact of the different gemini surfactants on the permeability and morphology of model vesicles was additionally investigated by measuring the leakage of calcein fluorescent dye and analyzing the NMR spectra of 31P, respectively. Detail on the interaction of gemini molecules with model membranes was also provided by a systematic differential scanning calorimetry (DSC) and molecular dynamics (MD) simulation. An irreversible impact on the viability of the NCTC 2544 cell line was observed for gemini concentrations higher than 25 mM, while no cytotoxicity was found for any of the surfactants in a concentration range up to 10 mM. A higher cytotoxicity was also found for gemini surfactants presenting longer spacer and shorter tails. The same trend was obtained in the calorimetric and permeability studies, with the gemini of longest spacer promoting the highest degree of membrane destabilization. Additional structural and dynamical characterization of the various systems, obtained by 31P NMR and MD, provide some insight on the relationship between the architecture of gemini surfactants and the respective perturbation mechanism

New micellar morphologies from amphiphilic block copolymers: disks, toroids and bicontinuous micelles

Amphiphilic AB and ABA block copolymers have been demonstrated to form a variety of self-assembled aggregate structures in dilute solutions where the solvent preferentially solvates one of the blocks. The most common structures formed by these amphiphilic macromolecules are spherical micelles, cylindrical micelles and vesicles (polymersomes). Interest into the characterisation and controlled formation of block copolymer aggregates has been spurred on by their potential as surfactants, nano- to micro-sized carriers for active compounds, for the controlled release of encapsulated compounds and for inorganic materials templating, amongst numerous other proposed applications. Research in the past decade has focussed not only on manipulating the properties of aggregates through control of both the chemistry of the constituent polymer blocks but also the external and internal morphology of the aggregates. This review article will present an overview of recent approaches to controlling the self-assembly of amphiphilic block copolymers with a view to obtaining novel micellar morphologies. Whilst the article touches upon multi-compartment micelles particular focus is placed upon control of the overall shape of micelles; i.e. those systems that expand the range of accessible morphologies beyond ‘simple’ spherical and cylindrical micelles namely disk-like, toroidal and bicontinuous micelles

Linkage identity is a major factor in determining the effect of PEG-ylated surfactants on permeability of phosphatidylcholine liposomes

The permeability effects induced by single-chained and double-chained poly(ethylene glycol)-surfactants were investigated by measuring the leakage of the fluorescent dye 5(6)-carboxy fluorescein from EPC liposomes. The standard incorporated amount of the surfactants was 5 mol%. Depending on the size of the poly(ethylene glycol) chain and especially on the type of linkage between the polymer and the hydrophobic moiety different leakage profiles were obtained. The presence of a long PEG-polymer resulted in a slower leakage compared with a short analogue. More importantly, the linkage identity was decisive for whether an overall reduction or increase in permeability was obtained. When the hydrocarbon chains were attached to the PEG chain via an ether or an ester the leakage increased compared to pure EPC liposomes. In contrast, if the link was an amide, the leakage was significantly reduced. This effect is assumed to originate from headgroup–headgroup interactions, and most probably hydrogen bonding, between amide and phosphate groups of the PEG-surfactant and the EPC, respectively</p

Effect of polyethyleneglycol-phospholipids on aggregate structure in preparations of small unilamellar liposomes

Phospholipids with covalently attached poly(ethylene glycol) (PEG lipids) are commonly used for the preparation of long circulating liposomes. Although it is well known that lipid/PEG-lipid mixed micelles may form above a certain critical concentration of PEG-lipid, little is known about the effects of PEG-lipids on liposome structure and leakage at submicellar concentrations. In this study we have used cryogenic transmission electron microscopy to investigate the effect of PEG(2000)-PE on aggregate structure in preparations of liposomes with different membrane compositions. The results reveal a number of important aggregate structures not documented before. The micrographs show that enclosure of PEG-PE induces the formation of open bilayer discs at concentrations well below those where mixed micelles begin to form. The maximum concentration of PEG-lipid that may be incorporated without alteration of the liposome structure depends on the phospholipid chain length, whereas phospholipid saturation or the presence of cholesterol has little or no effect. The presence of cholesterol does, however, affect the shape of the mixed micelles formed at high concentrations of PEG-lipid. Threadlike micelles form in the absence of cholesterol but adapt a globular shape when cholesterol is present

Rheological properties of phospholipid-stabilized parenteral oil-in-water emulsions - effects of electrolyte concentration and presence of heparin

The theological properties of the parenteral oil-in-water emulsion Intralipid(TM) were investigated. The viscosity data at different phase volumes correlated well with that obtained via a theoretical model developed by Yaron and Gal-Or. The model also describes the temperature dependence well. The effects of electrolyte addition were also investigated. Monovalent sodium chloride had practically no influence on viscosity. Calcium chloride, on the other hand, had a large impact on viscosity even at low concentrations. It was shown that the obtained maximum in viscosity coincided with the zeta-potential being close to zero. The resulting increase in viscosity is due to flocculation that leads to an increase in apparent phase volume. A similar behaviour was obtained with magnesium chloride with the difference that the maximum in viscosity was shifted to higher electrolyte concentrations. This is interpreted as that because magnesium binds strongly to the hydration water than does calcium. The addition of the negatively charged anti-coagulant heparin causes flocculation in the presence of small amounts of calcium. The amounts of calcium needed for such bridging flocculation is lower than what is needed in order to create a positive potential at the surfaces of the droplets. A fraction of the floes is not broken down even by extensive shear</p

Decreased levels of keratin 8 sensitize mice to streptozotocin-induced diabetes

AimDiabetes is a result of an interplay between genetic, environmental and lifestyle factors. Keratin intermediate filaments are stress proteins in epithelial cells, and keratin mutations predispose to several human diseases. However, the involvement of keratins in diabetes is not well known. K8 and its partner K18 are the main β-cell keratins, and knockout of K8 (K8−/−) in mice causes mislocalization of glucose transporter 2, mitochondrial defects, reduced insulin content and altered systemic glucose/insulin control. We hypothesize that K8/K18 offer protection during β-cell stress and that decreased K8 levels contribute to diabetes susceptibility.MethodsK8-heterozygous knockout (K8+/−) and wild-type (K8+/+) mice were used to evaluate the influence of keratin levels on endocrine pancreatic function and diabetes development under basal conditions and after T1D streptozotocin (STZ)-induced β-cell stress and T2D high-fat diet (HFD).ResultsMurine K8+/− endocrine islets express ~50% less K8/K18 compared with K8+/+. The decreased keratin levels have little impact on basal systemic glucose/insulin regulation, β-cell health or insulin levels. Diabetes incidence and blood glucose levels are significantly higher in K8+/− mice after low-dose/chronic STZ treatment, and STZ causes more β-cell damage and polyuria in K8+/− compared with K8+/+. K8 appears upregulated 5 weeks after STZ treatment in K8+/+ islets but not in K8+/−. K8+/− mice showed no major susceptibility risk to HFD compared to K8+/+.ConclusionPartial K8 deficiency reduces β-cell stress tolerance and aggravates diabetes development in response to STZ, while there is no major susceptibility to HFD