A5073809598- Advanced Fluorescence Microscopy Techniques
- RNA Interference and Gene Delivery
- Lipid Membrane Structure and Behavior
- Advanced biosensing and bioanalysis techniques
- Pancreatic function and diabetes
- Nanoparticle-Based Drug Delivery
- RNA Research and Splicing
- Diabetes and associated disorders
- Nuclear Structure and Function
- Diabetes Management and Research
- Spectroscopy and Quantum Chemical Studies
- Virus-based gene therapy research
- Immunotherapy and Immune Responses
- Cellular transport and secretion
- Photosynthetic Processes and Mechanisms
- Nanoplatforms for cancer theranostics
- Cancer Research and Treatments
- Genomics and Chromatin Dynamics
- Photoreceptor and optogenetics research
- Graphene and Nanomaterials Applications
- Advanced Electron Microscopy Techniques and Applications
- HIV Research and Treatment
- Luminescence and Fluorescent Materials
- Spectroscopy Techniques in Biomedical and Chemical Research
- Metabolism, Diabetes, and Cancer
Scuola Normale Superiore
2016-2025
National Enterprise for NanoScience and NanoTechnology
2009-2025
Istituto Nanoscienze
2017-2025
University of Pisa
2024
Center for Nanotechnology Innovation
2009-2017
Italian Institute of Technology
2006-2017
University of California, Irvine
2010-2012
Fluor (United States)
2010-2012
Fluorescence Innovations (United States)
2010
Istituto Nazionale per la Fisica della Materia
2009
Abstract The translational motion of molecules in cells deviates from what is observed dilute solutions. Theoretical models provide explanations for this effect but with predictions that drastically depend on the nanoscale organization assumed macromolecular crowding agents. A conclusive test nature missing owing to lack techniques capable probing required temporal and spatial resolution. Here we show fluorescence-fluctuation analysis raster scans at variable timescales can information. By...
Abstract Lipofectamine reagents are widely accepted as “gold-standard” for the safe delivery of exogenous DNA or RNA into cells. Despite this, a satisfactory mechanism-based explanation their superior efficacy has remained mostly elusive thus far. Here we apply straightforward combination live cell imaging, single-particle tracking microscopy and quantitative transfection-efficiency assays on cells to unveil intracellular trafficking mechanism Lipofectamine/DNA complexes. We find that...
Spatial distribution and dynamics of plasma-membrane proteins are thought to be modulated by lipid composition the underlying cytoskeleton, which forms transient barriers diffusion. So far this idea was probed single-particle tracking membrane components in gold particles or antibodies were used individually monitor molecules interest. Unfortunately, relatively large needed for can principle alter very under study. Here, we use a method that makes it possible investigate means small...
A possible turning point in drug delivery has been recently reached: the protein shell, which covers nanocarriers vivo, can be used for targeting. Here, we show that nanoparticles acquire a selective targeting capability with corona adsorbed on surface. We demonstrate lipid particles made of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and DNA, upon interaction human plasma components, spontaneously become coated vitronectin promotes efficient uptake cancer cells expressing high levels...
The biomolecular corona promotes a switch of both the cell entry mechanism and intracellular dynamics liposomes.
For several decades, surface grafted polyethylene glycol (PEG) has been a go-to strategy for preserving the synthetic identity of liposomes in physiological milieu and preventing clearance by immune cells. However, limited clinical translation PEGylated is mainly due to protein corona formation subsequent modification liposomes' identity, which affects their interactions with cells blood residency. Here we exploit electric charge DNA generate unPEGylated liposome/DNA complexes that, upon...
Here we investigate the cellular uptake mechanism and final intracellular fate of two cationic liposome formulations characterized by similar physicochemical properties but very different lipid composition efficiency for delivery DNA. The first formulation is made 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) zwitterionic helper dioleoylphosphocholine (DOPC), while second one 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) dioleoylphosphatidylethanolamine (DOPE)....
No methods proposed thus far have the capability to measure overall molecular flow in nucleus of living cells. Here, we apply pair correlation function analysis (pCF) anisotropic diffusion interphase live In pCF method, cross-correlate fluctuations at several distances and locations within nucleus, enabling us define migration paths barriers diffusion. We use monomeric EGFP as a prototypical inert molecule between different nuclear environments. Our results suggest that there are two...
Abstract The observation of molecular diffusion at different spatial scales, and in particular below the optical diffraction limit (<200 nm), can reveal details subcellular topology its functional organization. Stimulated-emission depletion microscopy (STED) has been previously combined with fluorescence correlation spectroscopy (FCS) to investigate nanoscale (STED-FCS). However, stimulated-emission only used successfully organization two-dimensional space, such as plasma membrane, while,...
More than 20 years after its approval by the Food and Drug Administration (FDA), liposomal doxorubicin (DOX) is still drug of choice for treatment breast cancer other conditions such as ovarian multiple myeloma. Yet, despite efforts, DOX did not satisfy expectations at clinical level. When drugs enter a physiological environment, their surface gets coated dynamic biomolecular corona (BC). The BC changes liposome's synthetic identity, providing it with new one, referred to "biological...
Abstract Loss of pancreatic beta cells is a central feature type 1 (T1D) and 2 (T2D) diabetes, but therapeutic strategy to preserve cell mass remains be established. Here we show that the death receptor TMEM219 expressed on signaling through its ligand insulin-like growth factor binding protein 3 (IGFBP3) leads loss dysfunction. Increased peripheral IGFBP3 was observed in established at-risk T1D/T2D patients confirmed preclinical models, suggesting dysfunctional IGFBP3/TMEM219 associated...
The extracellular signal-regulated protein kinase ERK1/2 is a crucial effector linking stimuli to cellular responses: upon phosphorylation ERK [also known as mitogen-activated P42/P44 (MAPK)] concentrates in the nucleus where it activates specific programs of gene expression. Notwithstanding importance this process, little about modalities, time course and regulation exchange between cytoplasm living cells. We visualized dynamic nuclear translocation by expressing low levels (<150 nM)...
Tat-derived peptides have attracted much interest as molecular carriers for intracellular delivery they incorporate specific attributes required efficient cargo to sub-cellular domains. Little is known, however, about trafficking and interactions of Tat peptide–tagged cargoes, although some in vitro studies suggested the relevance active processes peptide–driven nuclear translocation. These issues are addressed by comparing peptide–induced transport properties with well-established passive...
Background Nuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoproteins across the double-membrane nuclear envelope. Although there are many studies that look at traffic in nucleus through envelope we propose a method to detect nucleocytoplasmic kinetics an unperturbed cell, with no requirement for specific labeling isolated molecules and, most important, presence cell milieu. Methodology The pair correlation function (pCF) measures time molecule...
DNA vaccination has been extensively studied as a promising strategy for tumor treatment. Despite the efforts, therapeutic efficacy of vaccines limited by their intrinsic poor cellular internalization. Electroporation, which is based on application controlled electric field to enhance penetration into cells, method choice produce acceptable levels gene transfer in vivo. However, this may cause cell damage or rupture, non-specific targeting, and even degradation pDNA. Skin irritation, muscle...
Glioblastoma (GB) is the most severe form of brain cancer, with a 12-15 month median survival. Surgical resection, temozolomide (TMZ) treatment, and radiotherapy remain primary therapeutic options for GB, no new therapies have been introduced in recent years. This standstill primarily due to preclinical approaches that do not fully respect complexity GB cell biology fail test efficiently anti-cancer treatments. Therefore, better treatment screening are needed. In this study, we developed...
The supramolecular organization of Doxorubicin (DOX) within the standard Doxoves® liposomal formulation (DOX®) is investigated using visible light and phasor approach to fluorescence lifetime imaging (phasor-FLIM). First, phasor-FLIM signature DOX® resolved into contribution three co-existing fluorescent species, each with its characteristic mono-exponential lifetime, namely: crystallized DOX (DOXc, 0.2 ns), free (DOXf, 1.0 bound membrane (DOXb, 4.5 ns). Then, exact molar fractions species...
Formation and characterization of the protein corona on polystyrene nanoplastics its impact cytotoxicity, cellular uptake, signaling pathways in breast cancer cells.
Reversibly photoswitchable (i.e., photochromic) fluorescent proteins open the way to a number of advanced bioimaging techniques applicable living-cell studies such as sequential photolabeling distinct cellular regions, innovative FRET schemes, or nanoscopy. Owing relevance from Aequorea victoria (AFPs) for cell biology, photochromic "toolbox" constituted by several AFPs is highly desirable. Here we introduce four new whose reversible photoswitching occurs between native bright and dark state...
We packaged condensed DNA/protamine particles in multicomponent envelope-type nanoparticle systems (MENS) combining different molar fractions of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) zwitterionic dioleoylphosphocholine (DOPC) dioleoylphosphatidylethanolamine (DOPE). Dynamic light scattering (DLS) microelectrophoresis allowed us to identify lipid/DNA charge ratio at which MENS are small sized...
Abstract Lipid lateral diffusion in membrane bilayers is a fundamental process exploited by cells to enable complex protein structural and dynamic reorganizations. For its importance, lipid mobility both cellular model has been extensively investigated recent years, especially through the application of time-resolved, fluorescence-based, optical microscopy techniques. However, one caveat fluorescence techniques need use dye-labeled variants interest, thus potentially perturbing properties...