Quantitative Investigation by Atomic Force Microscopy of Supported Phospholipid Layers and Nanostructures on Cholesterol-Functionalized Glass Surfaces

Indrieri, Marco; Suardi, Marco; Podestà, Alessandro; Ranucci, Elisabetta; Ferruti, Paolo; Milani, Paolo

doi:10.1021/la703725b

Cited by 8 publications

(6 citation statements)

References 45 publications

(103 reference statements)

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“…Since the mechanical parameters derived from the dynamometer extrusion method are not absolute parameters, the results obtained with this approach were compared to those of the effective Young's modulus (log 10 (YM)) of single vesicles obtained by AFM nanoindentation tests. AFM analyses were performed in parallel since this technique has been widely employed for the topographic, physicochemical, and technological analysis of lipid layers and liposomes 26,34,35 and can effectively and accurately characterize the elastomechanical properties of nanoscale vesicles. 36 It was observed that the log 10 (YM) calculated by AFM followed the same trend of the resistance registered during the extrusion of the prepared liposomes suspensions, being higher for Fp populations than the corresponding Fs populations and F(−) (Table 3).…”

Section: Molecular Pharmaceuticsmentioning

confidence: 99%

Tuning the Extent and Depth of Penetration of Flexible Liposomes in Human Skin

et al. 2017

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In this work we made an attempt to assess the effect of drug-induced changes of flexibility on the penetration of deformable vesicles into the human skin. Eight cationic liposomes with different degrees of flexibility were obtained by entrapping unfractionated heparin, enoxaparin, and nadroparin. The deformability was studied by a novel, facile, and reliable extrusion assay appositely developed and validated by means of quantitative nanoscale mechanical AFM measurements of vesicle elastic modulus (log(YM)). The proposed extrusion assay, determining the forces involved in vesicles deformation, resulted very sensitive to evidence of minimal changes in bilayer rigidity (σ) and vesicle deformation (K). The drug loading caused a reduction of liposome flexibility with respect to the reference plain liposomes and in accordance to the heparin type, drug to cationic lipid (DOTAP) ratio, and drug distribution within the vesicles. Interestingly, the σ and log(YM) values perfectly correlated (R = 0.935), demonstrating the reliability of the deformability data obtained with both approaches. The combination of TEM and LC-MS/MS spectrometry allowed the pattern of the penetration of the entire vesicles into the skin to be followed. In all cases, intact liposomes in the epidermis layers were observed and a relationship between the depth of penetration and the liposome flexibility was found, supporting the hypothesis of the whole vesicle penetration mechanism. Moreover, the results of the extent (R) of vesicle penetration in the human skin samples showed a direct relation to the flexibility values (σ = 0.65 ± 0.10 MPa → R = 3.33 ± 0.02 μg/mg; σ = 0.95 ± 0.04 MPa → R = 1.18 ± 0.26 μg/mg; σ = 1.89 ± 0.30 MPa → R = 0.53 ± 0.33 μg/mg).

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Section: Molecular Pharmaceuticsmentioning

confidence: 99%

Tuning the Extent and Depth of Penetration of Flexible Liposomes in Human Skin

et al. 2017

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“…This makes SNOM a surface selective technique and SNOM reflection images can provide optical information about a thin layer below the cell surface. For such features, together with their high sensitivity and non‐invasiveness, they appear to be particularly suitable for investigating phenomena occurring at the level of plasma membrane (Rasmussen et al ., 2005; Indrieri et al , 2008; Müller, 2008; Jung et al ., 2010) and for visualizing sub‐membrane structures of whole cells with a flat morphology (Trevisan et al ., 2010). In this framework, we combined SEM and TEM with nanoscopic techniques as SNOM and AFM to obtain high‐resolution morphological description about the interaction of crocidolite fibres with mesothelial cells.…”

Section: Introductionmentioning

confidence: 99%

The crocidolite fibres interaction with human mesothelial cells as investigated by combining electron microscopy, atomic force and scanning near‐field optical microscopy

Andolfi

Trevisan

Zweyer

et al. 2013

Journal of Microscopy

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SummaryIn this study, we have performed a morphological analysis of crocidolite fibres interaction with mesothelial cells (MET5A) by combining conventional electron microscopy with atomic force (AFM) and scanning near-field optical microscopy (SNOM). After 6-h exposure at a crocidolite dose of 5 μg cm −2 , 90% of MET5A cells interact with fibres that under these conditions have a low cytotoxic effect. SEM images point out that fibres can be either engulfed by the cells that lose their typical morphology or they can accumulate over or partially inside the cells, which preserve their typical spread morphology. By using AFM we are able to directly visualize the entry-site of nanometric-sized fibres at the plasma membrane of the spread mesothelial cells. More importantly, the crocidolite fibres that are observed to penetrate the plasma membrane in SNOM topography can be simultaneously followed beneath the cell surface in the SNOM optical images. The analysis of SNOM data demonstrates the entrance of crocidolite fibres in proximity of nuclear compartment, as observed also in the TEM images. Our findings indicate that the combination of conventional electron microscopy with novel nanoscopic techniques can be considered a promising approach to achieve a comprehensive morphological description of the interaction between asbestos fibres and mesothelial cells that represents the early event in fibre pathogenesis.

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“…The dried lipids (0.1 mg) were rehydrated in PBS (phosphate buffer saline) maintaining 0.5 mg/ml concentration, the final solution increases turbidity due to formation of micelles. The ionic strength of PBS is necessary to shield effectively the electrostatic repulsive interaction between the negatively charged mica surface and phospholipids heads ( Reviakine and Brisson, 2000 ; Richter and Brisson, 2005 ; Indrieri et al, 2008 ). The lipids solution is vortexed and sonicated for 10 min.…”

Section: Methodsmentioning

confidence: 99%

Unveiling a Hidden Event in Fluorescence Correlative Microscopy by AFM Nanomechanical Analysis

Zhang

Wang

et al. 2021

Front. Mol. Biosci.

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Fluorescent imaging combined with atomic force microscopy (AFM), namely AFM-fluorescence correlative microscopy, is a popular technology in life science. However, the influence of involved fluorophores on obtained mechanical information is normally underestimated, and such subtle changes are still challenging to detect. Herein, we combined AFM with laser light excitation to perform a mechanical quantitative analysis of a model membrane system labeled with a commonly used fluorophore. Mechanical quantification was additionally validated by finite element simulations. Upon staining, we noticed fluorophores forming a diffuse weakly organized overlayer on phospholipid supported membrane, easily detected by AFM mechanics. The laser was found to cause a degradation of mechanical stability of the membrane synergically with presence of fluorophore. In particular, a 30 min laser irradiation, with intensity similar to that in typical confocal scanning microscopy experiment, was found to result in a ∼40% decrease in the breakthrough force of the stained phospholipid bilayer along with a ∼30% reduction in its apparent elastic modulus. The findings highlight the significance of analytical power provided by AFM, which will allow us to “see” the “unseen” in correlative microscopy, as well as the necessity to consider photothermal effects when using fluorescent dyes to investigate, for example, the deformability and permeability of phospholipid membranes.

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Quantitative Investigation by Atomic Force Microscopy of Supported Phospholipid Layers and Nanostructures on Cholesterol-Functionalized Glass Surfaces

Cited by 8 publications

References 45 publications

Tuning the Extent and Depth of Penetration of Flexible Liposomes in Human Skin

Tuning the Extent and Depth of Penetration of Flexible Liposomes in Human Skin

The crocidolite fibres interaction with human mesothelial cells as investigated by combining electron microscopy, atomic force and scanning near‐field optical microscopy

Unveiling a Hidden Event in Fluorescence Correlative Microscopy by AFM Nanomechanical Analysis

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