Tunable pDNA/DODAB:MO lipoplexes: the effect of incubation temperature on pDNA/DODAB:MO lipoplexes structure and transfection efficiency.

Dioctadecyldimethylammonium bromide (DODAB):1-monooleoyl-rac-glycerol (MO) cationic liposomes were reported as a promising alternative to common transfection agents, showing superior effectiveness on the transfection of the 293T mammalian cell line with pSV-ß-gal plasmid DNA. The study of DODAB:MO aggregates in the absence of DNA has indicated that their morphology depends on the balance between DODAB's tendency to form bilayer structures and MO's propensity to form inverted non-lamellar structures. Other parameters, such as the temperature have proved to be crucial in the definition of the morphology of the developed nanocarrier. Therefore, in this work, a step forward to the current gene carrier system will be given by studying the effect of the tunable parameters (incubation temperature and MO content) on the structure of pDNA:DODAB:MO lipoplexes. More importantly, the implications that these tunable parameters could have in terms of lipoplex transfection efficiency will be investigated. Dynamic light scattering (DLS), zeta (ζ) potential, cryo-transmission electron microscopy (cryo-TEM) and ethidium bromide (EtBr) exclusion were used to assess the formation, structure and destabilization of pDNA:DODAB:MO lipoplexes at DODAB molar fractions of (1:1) and above equimolarity (2:1, 4:1) prepared at incubation temperatures from 25 to 50°C. Experimental results indicate that pDNA:DODAB:MO's structure is sensitive to the lipoplex incubation temperature, resulting in particles of distinct size, superficial charge and structure. These variations are also visible on the complexation dynamics of pDNA, and subsequent release upon incubation with the model proteoglycan heparin (HEP), at 25 and 50°C. Increase in temperature leads to re-organization of DODAB and MO molecules within the liposomal formulation, causing a positive charge re-localization in the lipoplex surface, which not only alters its structure but also its transfection efficiency. Altogether, these results confirm that in the DODAB:MO carriers, an increase in the incubation temperature has a similar effect on aggregate morphology as the observed with an increase in MO content. This conclusion is extended to the pDNA:DODAB:MO lipoplexes morphology and subsequent transfection efficiency defining new strategies in lipoplexes preparation that could be used to modulate the properties of other lipid formulations for nonviral gene delivery applications.

Comparison of the tear film clinical parameters at two different times of the day.

PURPOSE: The aim of the present study was to analyse the quality and quantity of the tear film in a young, healthy and non-contact lens-wearing population with measurements taken in the morning and in the afternoon to establish if changes exist in these parameters. METHODS: In a controlled laboratory setting, morning and afternoon clinical parameters of tear film were assessed. Schirmer test, tear meniscus height, break-up time and non-invasive break-up time were measured in 51 normal subjects on the same day for each individual. RESULTS: Tear volume showed no significant changes during the day but tear film stability was significantly reduced at the end of the day (p < 0.001), demonstrating that tear film stability is affected by the time of day. CONCLUSION: Researchers and clinicians might wish to consider these differences when comparisons are made between studies and populations and also when the same patient is observed over periods. Fluorescein instillation can affect the measurements of tear stability in subjects with poor quality tear film.

Lens material and formulation of multipurpose solutions affects contact lens disinfection.

PURPOSE: To assess the disinfection efficacy of multipurpose solutions (MPS) against different bacterial species adhered either to silicon hydrogel or to conventional hydrogel contact lenses (CLs). The influences of the MPS formulation and the chemical composition of the lens material were investigated. METHODS: This investigation followed the standard 14729, which establishes the guidelines for assessing CL disinfecting solutions. Two commercially available (Opti-Free(®) Express(®) and Renu(®) Multiplus) solutions and one recalled solution (Complete(®) MoisturePlus™) were used in this study. After disinfection, the number of survivors was estimated by the colony forming units' method. RESULTS: The lens material appears to influence disinfection. The conventional hydrogel polymacon exhibited the highest disinfection scores, a fact that should be related with the lack of electrostatic attraction towards the biocides and its hydrophilicity. The MPS formulation appears to have influence in disinfection efficacy as well. For most adhered bacteria, Opti-Free(®) was capable of reducing cell concentration in 4-log. CONCLUSION: Disinfection results from multivariate factors and this study confirmed that the lens material and the MPS play a very important role in the disinfection efficacy of CL.

Contact lens hydrophobicity and roughness effects on bacterial adhesion.

PURPOSE: This study was designed to assess whether hydrogel contact lens (CL) surface hydrophobicity and roughness affect Staphylococcus epidermidis adhesion. METHODS: Bacterial adhesion experiments were performed on two unworn silicone hydrogel and three unworn conventional hydrogel CLs using the S.epidermidis strain CECT 4184. Microbial colonization was assessed by conducting counts expressed as colony-forming units. CL hydrophobicity was determined through water contact angle measurements and the roughness parameters such as mean surface roughness (Ra), kurtosis (Rku), and skewness (Rsk) were determined through atomic force microscopy in Tapping Mode. RESULTS: The conventional CLs showed similar water contact angles (p > 0.05) and were classified as hydrophilic. The silicone hydrogel CLs yielded hydrophobic contact angles with no significant differences between them (p > 0.05). The lenses with the highest (nelfilcon A and ocufilcon B) or lowest (comfilcon A and omafilcon A) Ra values displayed a lesser or greater extent of spikiness of their surfaces, respectively. All lenses showed a predominance of peaks (Rsk > 0) over troughs. S. epidermidis adhered more to the hydrophobic CLs (p < 0.05). Omafilcon A and comfilcon A, which showed the lowest Ra values among the hydrophilic and hydrophobic lenses, respectively, returned the lowest bacterial adhesion scores (p < 0.05). CONCLUSIONS: Our results suggest that more hydrophobic CLs are more prone to S. epidermidis adhesion. Although the Ra appears to be related to S. epidermidis adhesion, the influence of Rku and Rsk on this variable remains unclear.

Comparative study of silicone-hydrogel contact lenses surfaces before and after wear using atomic force microscopy.

PURPOSE: The aim of this study was to analyze surface changes of three silicone-hydrogel contact lenses after daily wear. The lenses used in this study were balafilcon A, lotrafilcon B (both surface-treated), and galyfilcon A (non surface-treated). METHODS: To understand how and where proteins, lipids, and other contaminants change contact lenses, surface roughness was assessed through Atomic Force Microscopy Tapping Mode. Roughness parameters were Mean Surface roughness (R(a)), Mean-square-roughness (R(q)), and Maximum roughness (R(max)). The surface topography of unworn and worn lenses was also mapped in great detail. RESULTS: Contact lenses roughness parameters exhibited different values before and after wear and the surface appearance also changed. After wear, balafilcon A and galyfilcon A showed a significant increase on surface roughness parameters, being this increase more accentuated to galyfilcon A. In lotrafilcon B materials no significant changes were observed with wear. CONCLUSIONS: The present study suggests that surface treatment of silicone-hydrogel contact lenses can play a role in the prevention of a significant increase in roughness, and contribute to the better clinical tolerance of these lenses.

The influence of surface treatment on hydrophobicity, protein adsorption and microbial colonisation of silicone hydrogel contact lenses.

PURPOSE: To evaluate the influence of surface treatment of silicone-hydrogel CL on lens hydrophobicity, protein adsorption and microbial colonisation by studying several silicone hydrogel contact lenses (CL) with and without surface treatment. The lenses used in this study were Balafilcon A, Lotrafilcon A, Lotrafilcon B and Galyfilcon A. A conventional hydrogel CL (Etafilcon A) was also tested. METHODS: Hydrophobicity was determined through contact angle measurement using the advancing type technique on air. The type and quantity of proteins adsorbed were assessed through SDS-PAGE and fluorescence spectroscopy, respectively. Microbial colonisation was studied by removing the microbes from the lenses through sonication, and counting the colony-forming units on agar plates. RESULTS: Regarding hydrophobicity, both surface and non-surface-treated silicone hydrogel CL were found to be hydrophobic, and the conventional hydrogel CL was found to be hydrophilic. Concerning protein adsorption, different protein profiles were observed on the several lenses tested. Nevertheless, the presence of proteins with the same molecular weight as lysozyme and lactoferrin was common to all lenses, which is probably related to their abundance in tears. In terms of total protein adsorption, silicone hydrogel CL did not exhibit any differences between themselves. However, the conventional hydrogel Etafilcon A adsorbed a larger amount of proteins. Regarding microbial colonisation, Balafilcon A exhibited the greatest amount of colonising microbes, which can be due to its superior hydrophobicity and higher electron acceptor capacity. CONCLUSION: This study suggests that silicone hydrogel lenses adsorb a lower amount of proteins than the conventional hydrogel lenses and that this phenomenon is independent of the presence of surface treatment. Concerning microbial colonisation, the surface treated Balafilcon A, exhibited a greater propensity, a fact that may compromise the lens wearer's ocular health.

Equivalences between refractive index and equilibrium water content of conventional and silicone hydrogel soft contact lenses from automated and manual refractometry.

PURPOSE: The purpose of the present study was to develop mathematical relationships that allow obtaining equilibrium water content and refractive index of conventional and silicone hydrogel soft contact lenses from refractive index measures obtained with automated refractometry or equilibrium water content measures derived from manual refractometry, respectively. METHODS: Twelve HEMA-based hydrogels of different hydration and four siloxane-based polymers were assayed. A manual refractometer and a digital refractometer were used. Polynomial models obtained from the sucrose curves of equilibrium water content against refractive index and vice-versa were used either considering the whole range of sucrose concentrations (16-100% equilibrium water content) or a range confined to the equilibrium water content of current soft contact lenses (approximately 20-80% equilibrium water content). RESULTS: Values of equilibrium water content measured with the Atago N-2E and those derived from the refractive index measurement with CLR 12-70 by the applications of sucrose-based models displayed a strong linear correlation (r2 = 0.978). The same correlations were obtained when the models are applied to obtain refractive index values from the Atago N-2E and compared with those (values) given by the CLR 12-70 (r2 = 0.978). No significantly different results are obtained between models derived from the whole range of the sucrose solution or the model limited to the normal range of soft contact lens hydration. CONCLUSIONS: Present results will have implications for future experimental and clinical research regarding normal hydration and dehydration experiments with hydrogel polymers, and particularly in the field of contact lenses.

Domain formation in DODAB-cholesterol mixed systems monitored via Nile Red anisotropy.

The effect of the cholesterol (Ch) on liposomes composed of the cationic lipid dioctadecyldimethylammonium bromide (DODAB) was assessed by studying both the steady-state and time-resolved fluorescence anisotropy of the dye Nile Red. The information obtained combined with analysis of the steady-state emission and fluorescence lifetime of Nile Red (NR) for different cholesterol concentrations (5-50%) elucidated the presence of "condensed complexes" and cholesterol-rich domains in these mixed systems. The steady-state fluorescence spectra were decomposed into the sum of two lognormal emissions, emanating from two different states, and the effect of temperature on the anisotropy decay of Nile Red for different cholesterol concentrations was observed. At room temperature, the time-resolved anisotropy decays are indicative of NR being relatively immobile (manifest by a high r (infinity) value). At higher temperature, rotational times ca. 1 ns were obtained throughout and a trend in increasing hindrance was seen with increase of Ch content.