269 research outputs found
Quantitative measurement of intracellular transport of nanocarriers by spatio-temporal image correlation spectroscopy
No full textSpatio-temporal image correlation spectroscopy (STICS) is a powerful technique for assessing the nature of particle motion in complex systems although it has been rarely used to investigate the intracellular dynamics of nanocarriers so far. Here we introduce a method to characterize the mode of motion of nanocarriers and to quantify their transport parameters on different length scales from single-cell to subcellular level. Using this strategy we were able to study the mechanisms responsible for the intracellular transport of DOTAP-DOPC/DNA and DC-Chol-DOPE/DNA lipoplexes in CHO-K1 live cells. Measurement of both diffusion coefficients and velocity vectors (magnitude and direction) averaged over regions of the cell revealed the presence of distinct modes of motion. Lipoplexes diffused slowly on the cell surface (diffusion coefficient, D ≈ 0.003 μm(2)/s). In the cytosol, the lipoplexes' motion was characterized by active transport with average velocity ν ≈ 0.03 μm/s and random motion. The method permitted us to generate intracellular transport map showing several regions of concerted motion of lipoplexes
An apolipoprotein-enriched biomolecular corona switches the cellular uptake mechanism and trafficking pathway of lipid nanoparticles
No full textFollowing exposure to biological milieus (e.g. after systemic administration), nanoparticles (NPs) get covered by an outer biomolecular corona (BC) that defines many of their biological outcomes, such as the elicited immune response, biodistribution, and targeting abilities. In spite of this, the role of BC in regulating the cellular uptake and the subcellular trafficking properties of NPs has remained elusive. Here, we tackle this issue by employing multicomponent (MC) lipid NPs, human plasma (HP) and HeLa cells as models for nanoformulations, biological fluids, and target cells, respectively. By conducting confocal fluorescence microscopy experiments and image correlation analyses, we quantitatively demonstrate that the BC promotes a neat switch of the cell entry mechanism and subsequent intracellular trafficking, from macropinocytosis to clathrin-dependent endocytosis. Nano-liquid chromatography tandem mass spectrometry identifies apolipoproteins as the most abundant components of the BC tested here. Interestingly, this class of proteins target the LDL receptors, which are overexpressed in clathrin-enriched membrane domains. Our results highlight the crucial role of BC as an intrinsic trigger of specific NP–cell interactions and biological responses and set the basis for a rational exploitation of the BC for targeted delivery
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Oligomerization of Concanavalin A in live cells detected by fluctuation analysis.
No full textMechanistic evaluation of the transfection barriers involved in lipid-mediated gene delivery: Interplay between nanostructure and composition
No full textHere we present a quantitative mechanism-based investigation aimed at comparing the cell uptake, intracellular trafficking, endosomal escape and final fate of lipoplexes and lipid-protamine/deoxyribonucleic acid (DNA) (LPD) nanoparticles (NPs) in living Chinese hamster ovary (CHO) cells. As a model, two lipid formulations were used for comparison. The first formulation is made of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic lipid dioleoylphosphocholine (DOPC), while the second mixture is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE). Our findings indicate that lipoplexes are efficiently taken up through fluid-phase macropinocytosis, while a less efficient uptake of LPD NPs occurs through a combination of both macropinocytosis and clathrin-dependent pathways. Inside the cell, both lipoplexes and LPD NPs are actively transported towards the cell nucleus, as quantitatively addressed by spatio-temporal image correlation spectroscopy (STICS). For each lipid formulation, LPD NPs escape from endosomes more efficiently than lipoplexes. When cells were treated with DOTAP-DOPC-containing systems the majority of the DNA was trapped in the lysosome compartment, suggesting that extensive lysosomal degradation was the rate-limiting factors in DOTAP-DOPC-mediated transfection. On the other side, escape from endosomes is large for DC-Chol-DOPE-containing systems most likely due to DOPE and cholesterol-like molecules, which are able to destabilize the endosomal membrane. The lipid-dependent and structure-dependent enhancement of transfection activity suggests that DNA is delivered to the nucleus synergistically: the process requires both the membrane-fusogenic activity of the nanocarrier envelope and the employment of lipid species with intrinsic endosomal rupture ability. © 2013 Elsevier B.V
Fluctuation Methods To Study Protein Aggregation in Live Cells: Concanavalin A Oligomers Formation
No full textPrefibrillar oligomers of proteins are suspected to be the primary pathogenic agents in several neurodegenerative diseases. A key approach for elucidating the pathogenic mechanisms is to probe the existence of oligomers directly in living cells. In this work, we were able to monitor the process of aggregation of Concanavalin A in live cells. We used number and brightness analysis, two-color cross number and brightness analysis, and Raster image correlation spectroscopy to obtain the number of molecules, aggregation state, and diffusion coefficient as a function of time and cell location. We observed that binding of Concanavalin A to the membrane and the formation of small aggregates paralleled cell morphology changes, indicating progressive cell compaction and death. Upon protein aggregation, we observed increased membrane water penetration as reported by Laurdan generalized polarization imaging
Novel Mechanisms of Cell Uptake in Lipid-Mediated Gene Delivery
No full textThe mechanism of cell uptake in lipid mediated gene delivery was investigated in NIH3T3 and CHO cell lines. We show that different endocytic pathways are activated by shape coupling between lipoplex and membrane lipids. Our results suggest that tailoring the lipoplex lipid composition to the patchwork-like plasma membrane profile could be a successful machinery of coordinating the endocytic pathway activities and the subsequent intracellular processing.
Transfection experiments performed at 4C, when endocytosis does not take place, show that a novel class of highly efficient multicomponent lipoplexes enter cells by a temperature-independent fusion-like mechanism. In vivo, plasma
proteins bind to lipoplex surface and create a rich ‘protein corona’ that is recognized by cells and other biological structures. The ‘protein corona’ associated to lipoplexes after interaction with human plasma was found to be much richer in basic immunoglobulins gamma proteins (Ig-Gs) than that of pure lipid
vesicles in the absence of DNA. Because surface properties of lipoplexes may determine their interaction with cells and tissues, an accurate knowledge of lipoplex surface properties may be important for predicting biological responses.
These findings also suggest the existence of hybrid structures made of multilamellar complexes either stuck together by DNA or coexisting with DNA loaded intact vesicles
Efficient escape from endosomes determines the superior efficiency of multicomponent lipoplexes.
No full textDesigner multicomponent lipoplexes have recently emerged as especially promising transfection candidates, since they are from 10 to 100 times more efficient than binary complexes usually employed for gene delivery purposes. Here, we show, for the first time, that after internalization binary complexes of lower transfection potency remain in compact perinuclear endosomes, while multicomponent systems have intrinsic endosomal rupture properties that allow plasmid DNA to escape from endosomes with extremely high efficiency. Endosomal rupture results in an extraordinarily homogeneous distribution of unbound plasmid DNA throughout the cytoplasm and in the nucleus
The Role of Ethics and Product Personality in the Intention to Purchase Organic Food Products: A Structural Equation Modelling Approach
No full textThe aim of the present study is to examine the role of ethical dimensions and product personality in the purchasing intention of organic food products. The Prospect method (Caprara, Barbaranelli and Guido 2000), which integrates the Five factors model of personality (FFM, cf. Digman 1990) and the Theory of planned behavior (TPB, Ajzen 1991) extended to an ethical dimension, was employed, by using a Structural Equation Modeling approach. Results showed that moral norms – i.e., personal beliefs regarding what is right or wrong (Parker, Manstead and Stradling 1995) – can be considered the main motivator of purchasing intention, and they are, in turn, affected by subjective norms and product personality traits of Naturalness and Authenticity. Marketing implications for firms operating in the organic food industry are discussed, in their intent to shift from a “niche” market to a broader diffusion of these products
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Amyloid-β(1-42) Peptide Induces Neutral Lipids Accumulation in Hippocampal Neurons in Normal and Alzheimer’s Disease Aging Models, Lipid Environment Remodeling Revealed by Hyperspectral Imaging and the Phasor Approach
No full textAmyloid-β (Aβ), particularly the Aβ(1-42) variant, plays dual roles in neuronal physiology and pathology. While essential for synaptic function at physiological levels, its accumulation induces oxidative stress, membrane disruption, and lipid peroxidation, contributing to Alzheimer’s disease (AD). Aβ interacts with lipid rafts, disturbing membrane composition and promoting further Aβ production. Lipid droplets (LDs), key in lipid storage, lipid transport and stress response, are altered by Aβ, showing impaired mobilization and increased abundance. This study uses nile red solvatochromic properties and hyperspectral imaging to analyze the lipid polarity of hippocampal neurons and LDs dynamics in neurons exposed to exogenous-labeled Aβ(1-42) (exAβ), revealing age- and genotype-dependent lipid remodeling in normal- and AD-aging.We employed hyperspectral confocal imaging and advanced image analysis to map lipid organization in primary hippocampal neurons from non-transgenic (NTg) and triple-transgenic (3xTg) AD mice in response to exAβ-mediated stress, using nile red, a lipophilic and environment sensitive fluorescent dye. The spectral phasor transformation converted each pixel’s emission spectrum into G and S coordinates, enabling unmixing of fluorescently tagged exAβ, and Nile Red’s neutral and polar lipid signals without fitting models. Custom segmentation generated binary masks for whole-cell, plasma membrane, cytosol, lipid droplets and vesicular structures containing exAβ (Ves-exAβ+), by thresholding channel projections and applying morphological operations. For spatial analysis, Phasor analysis of Local Image Correlation Spectroscopy (PLICS) computed local autocorrelation functions over m×m subimages, then applied Fourier phasor transforms to extract real size and clustering metrics of LDs and Ves-exAβ+. This combined pipeline quantified compartment-specific lipid polarity indices, droplet number, size distributions, and spatial relationships, revealing age and genotype-dependent lipid remodeling under exogenous Aβ(1-42) exposure.Hyperspectral confocal imaging with three-component spectral phasor analysis and PLICS revealed that both, genotype and exogenous Aβ(1-42), independently remodel lipid organization in hippocampal neurons. Our results link higher exAβ accumulation to lower lipid polarity, genotype-driven polarity differences and age-dependent increases in lipid droplet (LD) and Ves-exAβ+ counts. In whole-cell, cytosol, and membrane compartments, NTg neurons show steadily rising lipid polarity profiles from young to middle age, whereas 3xTgAD cells peak at middle age then decline, with exAβ accelerating polarity loss in both. LD analysis demonstrated that NTg LD counts rise then fall with age but are amplified by exAβ, with small LDs shrinking from ~81% to ~29% and large LDs enlarging to ~71%, while 3xTg-AD neurons exhibit early LD enlargement and altered clustering under exAβ stress. Finally, Ves-exAβ+ structures increase with age—more abruptly in 3xTgAD—cluster more tightly or fuse in old cells, and shift toward larger Aβ-rich vesicles, whose lipid polarity varies by size and genotype, highlighting complex, age and genotype-dependent Aβ handling.Together, these results demonstrate that exogenous Aβ(1-42) and AD genotype independently drive compartment-specific lipid remodeling, they jointly accelerate polarity loss in the membrane, cytosol, and LD pools, amplifying LD number, size, and clustering, and promoting the formation of larger, more hydrophobic Aβ-positive vesicles, highlighting dynamic lipid–Aβ codependency in aging neurons
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