Biology subjects

Albertazzi, L.

Publications and source records attributed to Albertazzi, L..

13 recordsLinked to original sources

bioRxiv2026

Mechanoscribed Xolography for Multiscale Mechanical Patterning of Cell-laden Hydrogels

Mechanotransduction is a key regulator of healthy and malignant tissue development. However, current mechanobiological insights are largely derived from cells cultured on two dimensional substrates or in homogeneous biomaterials, which insufficiently capture the three dimensional (3D) heterogeneity and dynamic nature of the native extracellular matrix. This gap persists due to a lack of manufacturing processes and analysis modalities to control and monitor material properties and their evolution in 3D. Here, Mechanoscribed Xolography is introduced as a light sheet volumetric bioprinting process to rapidly fabricate 3D cell laden hydrogels with spatially controlled mechanical heterogeneity across multiple length scales. Moreover, synchronizing in-situ optical microscopy with printing enables autonomous detection and spatial location driven printing around cellular spheroids. The refinement and spatiotemporal evolution of material properties are assessed with Brillouin microscopy, which verifies that the contrast of imprinted heterogeneity is preserved over three weeks. Remarkably, regional differences in cell behavior are attained within a one-step, one-material construct with elasticity of approximately 0.3 kPa to 0.8 kPa, or 0.7 kPa to 2.9 kPa with a crosslinking-enhancing additive. This foundational work on high-precision, rapid bioprinting surrounding cellular objects represents a unique tool to systematically advance experimentation of 3D mechanotransduction and prospectively engineer cell instructive biomaterials for regenerative medicine.

bioengineering↗

bioRxiv2026

Single-molecule imaging and tracking on clinical liquid biopsies reveals cancer biomarkers nanoscale organization and heterogeneity

Single-molecule imaging and tracking have revealed fundamental biological mechanisms at the molecular scale, yet their application to clinical research remains limited by technical complexity and sample preparation incompatible with patient-derived specimens. As a result, we lack information with molecular-scale resolution of clinically relevant biomarkers. Here, we develop a workflow enabling Points Accumulation in Nanoscale Topography (PAINT) combined with single-particle tracking (SPT) on clinical liquid biopsies, allowing analysis of biomarker nanoscale organization at the single-molecule level in cancer patients. Our approach features a sample preparation tailored to liquid biopsies and requires no fixation, covalent labelling, or genetic modification, making single-molecule imaging compatible with hospital clinical workflows. We demonstrate the methods diversity by imaging liquid biopsies from blood, bone marrow aspirates, and pleural effusions across different cancer types. PAINT-SPT captures both the expression and mobility of clinically relevant membrane receptor biomarkers, revealing pronounced inter- and intra-patient heterogeneity at the molecular and cellular levels. We discover that individual patients exhibit distinct molecular mobility fingerprints that reflect biomarker interaction states and correlate with clinical diagnostic readouts. Furthermore, these fingerprints distinguish healthy from cancer cells, enabling the development of a classifier that accurately identifies cancer cells based on their single-molecule behaviour. Together, our results establish a route to investigate patient-derived clinical samples at the single-molecule level and open new opportunities to understand cancer biology and biomarker function beyond ensemble-averaged measurements.

biophysics↗

bioRxiv2025

Automated and Simulation-Guided Multiplexed DNA-PAINT for Nanoparticle Characterization

Recent advances in lab automation have dramatically increased the throughput of material synthesis, enabling rapid screening of nanomaterials for specific applications in nanotechnology and nanomedicine. However, this progress highlights a key bottleneck: most high-resolution characterization techniques, such as electron microscopy, atomic force microscopy (AFM), and super-resolution microscopy, remain low-throughput and labor-intensive. To keep pace, characterization must evolve toward greater automation and scalability. Here, we present an integrated and automatable workflow for multicolor DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) super-resolution microscopy tailored for nanoparticles. Our approach combines kinetic simulations, automated multiplexed imaging, and streamlined image analysis to enable end-to-end automation, from experimental design to quantitative data output. Simulations predict optimal experimental conditions, thus reducing the need for manual optimization. A fluidics system paired with a TIRF microscope is used to automate multiplexed imaging by rounds of imaging and probe exchange (exchange-PAINT) on multiple memorized positions without human oversight during the acquisition process. Finally, an image analysis pipeline tailored for NPs allows for the quantification of nanoparticle size and multiplexed ligand functionalization. This methodology improves the throughput and reproducibility of single-molecule localization microscopy (SMLM) using DNA-PAINT and lowers the entry barrier for non-expert users, thus paving the way for broader adoption in nanomedicine and materials discovery workflows.

biophysics↗

bioRxiv2025

"Visualize, describe, compare" - nanoinformatics approaches for material-omics

Bioinformatics and cheminformatics are established disciplines, but nanoinformatics, the development of computational tools for understanding and designing nanomaterials, is still in its infancy. In light of the new data-driven approaches for nanomaterials discovery, there is a growing need for in silico tools tailored to analyze nanomaterials datasets. This is particularly crucial for soft materials, where a crystalline structure cannot be obtained and therefore the characterization datasets are less structured, and there are no standard methods for data mining. Here we present a computational package capable of visualizing, describing, and comparing nanoparticle datasets obtained with super-resolution microscopy at the single-particle and single-molecule level. Our method allows us to: i) visualize multiparametric nanoparticle datasets to grasp material properties and heterogeneity; ii) have a quantitative evaluation of a material through a series of molecular descriptors, and iii) compare different materials quantitatively and globally, going beyond comparison of a single property. We applied this method to a library of targeted nanoparticles revealing particle heterogeneity, similarities, and correlations between the synthesis and the physicochemical properties of the different nanomaterials. Finally, we show the potential of this approach to reveal batch-to-batch variations in time and between users hidden in standard analysis.

bioinformatics↗

bioRxiv2025

Glyco-PAINT-APP: Subcellular analysis of immune cell-lectin binding enables correlation of glycan binding parameters to receptor biology

Extracting single-molecule lectin binding kinetics from primary cells has not been possible to date. Here, we present Glyco-PAINT-APP (Automated Processing Pipeline), an automated method that enables the extraction of subcellular glycan interaction kinetics using a Points Accumulation for Imaging in Nano-Topography (PAINT)-based approach. This approach leverages an algorithm for precise, high-throughput subcellular analysis of glycan binding dynamics, facilitating the exploration of functional correlations between glycoform binding patterns and immune cell polarization. Using synthetic glycans and glycosylated antigens, we demonstrate the ability of the technique to automatically correlate glycan binding parameters in subregions of dendritic cell membranes with increased uptake and cross-presentation efficiency of these antigens. Additionally, we show how the method can uncover subtle differences in glycan binding preferences between resting and polarized macrophages. Taken together, Glyco-PAINT-APP has the potential to enable new insights into the cell-intrinsic heterogeneity of glycan-structure-activity relationships in immune cells.

cell biology↗

bioRxiv2024

Unveiling DNA Origami Interaction Dynamics on Living Cell Surfaces by Single Particle Tracking

Due to the unique spatial addressability of DNA origami, targeting ligands (e.g. aptamers or antibodies) can be specifically positioned onto the surface of the nanostructure, constituting an essential tool for studying ligand-receptor interactions at the cell surface. While the design and ligand incorporation into DNA origami nanostructures are well-established, the study of cell surface interaction dynamics is still in the explorative phase, where in depth fundamental understanding on the molecular interactions remains underexplored. This study uniquely captures real-time encounters between DNA origami and cells in-situ using single particle tracking (SPT). Here, we functionalized DNA nanorods (NRs) with antibodies or aptamers specific to the epidermal growth factor receptor (EGFR) and used them to target EGFR-overexpressing cancer cells. SPT data revealed that ligand coated NRs selectively bound to the receptors expressed in target cancer cells, while non-functionalized NRs only display negligible cell interactions. Furthermore, we explored the effect of ligand density on the DNA origami, which revealed that aptamer-decorated NRs exhibit non-linear binding characteristics, whereas this effect in antibody-decorated NRs was less pronounced. This study provides new mechanistic insights into the fundamental understanding of DNA origami behaviour at the cell interface, with unprecedented spatiotemporal resolution, aiding the rational design of ligand-targeted DNA origami for biomedical applications.

bioengineering↗

bioRxiv2024

nanoFeatures: a cross-platform application to characterize nanoparticles from super-resolution microscopy images

Super-resolution microscopy and Single-Molecule Localization Microscopy (SMLM) are a powerful tool to characterize synthetic nanomaterials used for many applications such as drug delivery. In the last decade, imaging techniques like STORM, PALM, and PAINT have been used to study nanoparticle size, structure, and composition. While imaging has progressed significantly, often image analysis did not follow accordingly and many studies are limited to qualitative and semi-quantitative analysis. Therefore, it is imperative to have a robust and accurate method to analyze SMLM images of nanoparticles and extract quantitative features from them. Here we introduce nanoFeatures, a cross-platform Matlab-based app for the automatic and quantitative analysis of super-resolution images. nanoFeatures makes use of clustering algorithms to identify nanoparticles from the raw data (localization list) and extract quantitative information about size, shape, and molecular abundance at the single-particle and single-molecule levels. Moreover, it applies a series of quality controls, increasing data quality and avoiding artifacts. nanoFeatures, thanks to its intuitive interface is also accessible to non-experts and will facilitate analysis of super-resolution microscopy for materials scientists and nanotechnologies. This easy accessibility to expansive feature characterization at the single particle level will bring us one step closer to understanding the relationship between nanostructure features and their efficiency. https://github.com/n4nlab/nanoFeatures

bioinformatics↗

bioRxiv2023

Reaching the tumor: mobility of polymeric micelles inside an in vitro tumor-on-a-chip model with dual ECM

Degradable polymeric micelles are promising drug delivery systems due to their hydrophobic core and responsive design. When applying micellar nanocarriers for tumor delivery, one of the bottlenecks encountered in vivo is the tumor tissue barrier - crossing the dense mesh of cells and extracellular matrix (ECM). Sometimes overlooked, the extracellular matrix can trap nanoformulations based on charge, size and hydrophobicity. Here we used a simple design of a microfluidic chip with two types of ECM and MCF7 spheroids to allow "high throughput" screening of the interactions between biological interfaces and polymeric micelles. To demostrarte the applicapility of the chip, a small library of fluorescently labelled polymeric micelles varying in their hydrophilic shell and hydrophobic core forming blocks was studied. Three widely used hydrophilic shells were tested and compared, poly(ethylene glycol), poly(2-ethyl-2-oxazoline), and poly(acrylic acid), along with two enzyamticaly degradable dendritic hydrophobic cores (based on Hexyl or Nonyl end-gorups). Using ratiometric imaging of unimer:micelle fluorescence and FRAP inside the chip model, we obtained the local assembly state and dynamics inside the chip. Notably, we observed different micelle behaviors in the basal lamina ECM, from avoidance of ECM structure to binding of the poly(acrylic acid) formulations. Binding to basal lamina correlated with higher uptake into MCF7 spheroids. Overall, we proposed a simple microfluidic chip containing dual ECM and spheroids for the assessment of the interactions of polymeric nanocarrires with biological interfaces and evaluating nanoformulations capacity to cross the tumor tissue barrier.

bioengineering↗

bioRxiv2022

Enabling spectrally resolved single-molecule localization microscopy at high emitter densities

Single-molecule localization microscopy (SMLM) is a powerful technique for elucidating structure and dynamics in the life- and material sciences with sub-50 nm spatial resolution. The simultaneous acquisition of spectral information (spectrally resolved SMLM, sSMLM) enables multiplexing using spectrally distinct fluorophores or enable the probing of local chemical environments by using solvachromatic fluorophores such as Nile Red. Until now, the widespread utilisation of sSMLM was hampered by several challenges: an increased complexity of the optical detection pathway, limited software solutions for data analysis, lower accessible emitter densities or smaller field-of-views, and overall compromised spatio-spectral resolution. Here, we present a low-cost implementation of sSMLM that addresses these challenges. Using a blazed, low-dispersion transmission grating positioned close to the image plane here represented by the camera sensor, the +1st diffraction order is minimally elongated compared to the point spread function of the 0th order and can therefore be analysed using common subpixel single-molecule localization algorithms. The distance between both PSFs provides accurate information on the spectral properties of the emitter. The minimal excess width of 1st order PSFs enables a fivefold higher emitter density compared to other sSMLM approaches whilst achieving a spatio-spectral localization accuracy sufficient to discriminate between fluorophores whose peak emission are less than 15 nm apart as demonstrated using dSTORM, DNA-PAINT and smFRET. We provide an ImageJ/Fiji plugin (sSMLMAnalyzer) and suitable Matlab scripts for data analysis. We envision that our approach will find widespread use in super-resolution applications that rely on distinguishing spectrally different fluorophores under low photon conditions.

biophysics↗

bioRxiv2021

Determinants of ligand-functionalized DNA nanostructure-cell interactions

Synthesis of ligand-functionalized nanomaterials with control over size, shape and ligand orientation, facilitates the design of tailored nanomedicines for therapeutic purposes. DNA nanotechnology has emerged as a powerful tool to rationally construct two- and three-dimensional nanostructures, enabling site-specific incorporation of protein ligands with control over stoichiometry and orientation. To efficiently target cell surface receptors, exploration of the parameters that modulate cellular accessibility of these nanostructures is essential. In this study we systematically investigate tunable design parameters of antibody-functionalized DNA nanostructures binding to therapeutically relevant receptors. We show that, although the native affinity of antibody-functionalized DNA nanostructures remains unaltered, the absolute number of bound surface receptors is lower compared to soluble antibodies and is mainly governed by nanostructure size and DNA handle location. The obtained results provide key insights in the ability of ligand-functionalized DNA nanostructures to bind surface receptors and yields design rules for optimal cellular targeting.

synthetic biology↗

bioRxiv2020

The ESCRT-III machinery participates in the production of extracellular vesicles and protein export during Plasmodium falciparum infection

Infection with Plasmodium falciparum enhances extracellular vesicles (EVs) production in parasitized red blood cells (pRBC), an important mechanism for parasite-to-parasite communication during the asexual intraerythrocytic life cycle. The endosomal sorting complex required for transport (ESCRT), and in particular the ESCRT-III sub-complex, participates in the formation of EVs in higher eukaryotes. However, RBCs have lost the majority of their organelles through the maturation process, including an important reduction in their vesicular network. Therefore, the mechanism of EV production in P. falciparum-infected RBCs remains to be elucidated. Here we demonstrate that P. falciparum possesses a functional ESCRT-III machinery that is activated by an alternative recruitment pathway involving the action of PfBro1 and PfVps32/PfVps60 proteins. Additionally, multivesicular bodies formation and membrane shedding, both reported mechanisms of EVs production, were reconstituted in the membrane model of giant unilamellar vesicles using the purified recombinant proteins. Moreover, the presence of PfVps32, PfVps60 and PfBro1 in EVs purified from a pRBC culture was confirmed by super-resolution microscopy. In accordance, disruption of the Pfvps60 gene led to a reduction in the number of the produced EVs in the KO strain when compared with the parental 3D7 strain. Overall, our results increase the knowledge on the underlying molecular mechanisms during malaria pathogenesis and demonstrate that ESCRT-III P. falciparum proteins participate in EVs production.

microbiology↗

bioRxiv2020

Real-time Ratiometric Imaging of Micelles Assembly State in a Microfluidic Cancer-on-a-chip

The performance of supramolecular nanocarriers as drug delivery systems depends on their stability in the complex and dynamic biological media. After administration, nanocarriers are challenged by confronting different barriers such as shear stress and proteins present in blood, endothelial wall, extracellular matrix and eventually cancer cell membranes. While early disassembly will result in a premature drug release, extreme stability of the nanocarriers can lead to poor drug release and low efficiency. Therefore, comprehensive understanding of the stability and assembly state of supramolecular carriers in each stage of delivery is a key factor for the rational design of these systems. One of the key challenges is that current 2D in vitro models do not provide exhaustive information, as they do not fully recapitulate the 3D tumor microenvironment. This deficiency of the 2D models complexity is the main reason for the differences observed in vivo when testing the performance of supramolecular nanocarriers. Herein, we present a real-time monitoring study of self-assembled micelles stability and extravasation, combining spectral confocal microscopy and a microfluidic tumor-on-a-chip. The combination of advanced imaging and a reliable organ-on-a-chip model allow us to track micelle disassembly by following the spectral properties of the amphiphiles in space and time during the crucial steps of drug delivery. The spectrally active micelles were introduced under flow and their position and conformation followed during the crossing of barriers by spectral imaging, revealing the interplay between carrier structure, micellar stability and extravasation. Integrating the ability of the micelles to change their fluorescent properties when disassembled, spectral confocal imaging and 3D microfluidic tumor blood vessel-on-a-chip, resulted in the establishment of a robust testing platform, suitable for real-time imaging and evaluation of supramolecular drug delivery carriers stability.

bioengineering↗

bioRxiv2019

Efficient small-scale conjugation of DNA to primary antibodies for multiplexed cellular targeting

The combination of the specificity of antibodies and the programmability of DNA nanotechnology has provided the scientific community with a powerful tool to label and unambiguously distinguish a large number of subcellular targets using fluorescence-based read-out methods. While primary antibodies are commercially available for a large class of targets, a general stoichiometric site-specific DNA labeling strategy for this affinity reagent is lacking. Here, we present a universal, site-selective, conjugation method using a small photocrosslinkable protein G adaptor that allows labeling of antibodies of different host species with a controlled number of short oligonucleotides (ODNs). Importantly, we illustrate that this conjugation method can be directly performed on commercially-available primary antibodies, on a small scale and without cross-reactivity towards other proteins, such as bovine serum albumin. In addition, we present a general, benchtop-compatible strategy to purify DNA-labeled antibodies without loss of function. The application of protein G-ODN labeled primary antibodies is demonstrated by employing three well-known methods for detecting subcellular targets using fluorescent read-out, including flow cytometry, DNA-PAINT, and dSTORM. This work thus establishes a general and efficient platform for the synthesis of a library of unique ODN-antibody conjugates, facilitating the broader use of DNA-based programmable tags for multiplexed labeling to identify subcellular features with nanometer-precision, improving our understanding of cellular structure and function.

biochemistry↗