Biology subjects

Huysecom, S.

Publications and source records attributed to Huysecom, S..

3 recordsLinked to original sources

bioRxiv2026

When lysosomes persist: resolving the proton-sponge paradox in nanoparticle-based intracellular delivery

Proton-sponge-active polymers are widely used in nanomedicine to enhance intracellular delivery, yet the mechanism by which they promote cytosolic release of therapeutic cargo remains under debate. Whether these materials drive complete endolysosomal escape or instead alter lysosomal integrity without full nanoparticle release remains unclear. Here we show that polyethylene imine (PEI), a prototypical proton sponge active polymer, induces lysosomal membrane destabilization rather than full nanoparticle escape. Using PEI-coated mesoporous silica nanoparticles as a model delivery system, we show that PEI promotes cytosolic release of small-molecule cargo while nanoparticles remain confined within membrane-enclosed LAMP1-positive compartments. This behaviour arises from the combination of partial lysosomal membrane permeabilization and lysosomal deacidification, which together enable cargo leakage while impairing detection of lysosomes by pH-dependent probes. Our results resolve a long-standing ambiguity in the nanomedicine field and provide a revised mechanistic framework for interpreting endolysosomal escape in intracellular delivery. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/721565v2_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@74b98org.highwire.dtl.DTLVardef@f405eborg.highwire.dtl.DTLVardef@b0a276org.highwire.dtl.DTLVardef@79f154_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Quantitative Analysis of Cytoplasmic Viscosity in Colorectal Cancer Cells by Differential Dynamic Microscopy of Genetically Encoded Nanoparticles

The viscosity of the cytoplasm plays a key role in regulating molecular diffusion and cellular mechanics, yet quantifying it in living cells remains technically challenging. Genetically encoded multimeric nanoparticles (GEMs) have emerged as powerful probes for intracellular microrheology; however, current analyses rely on single-particle tracking, which is limited by probe density, imaging noise, and expression variability. Here, we combine GEMs with differential dynamic microscopy (DDM) to enable quantitative, non-invasive, and rapid measurement of intracellular viscosity using standard wide-field fluorescence imaging. DDM extracts particle dynamics from ensemble spatiotemporal intensity fluctuations, yielding reliable diffusion coefficients and viscosity values even in crowded or heterogeneous environments where tracking fails. Validation with fluorescent nanoparticles diffusing in water confirmed that DDM accurately reproduced theoretical viscosities across a wide range of particle sizes and concentrations. Comparison with single-particle tracking (SPT) demonstrated equivalent precision under dilute conditions and superior robustness under crowding. To showcase the potential of this approach, we applied GEM-DDM to colorectal cancer cell lines with different metastatic potentials. Cytoplasmic viscosity correlated with aggressiveness, increasing from 1.9-2.3 cP in poorly metastatic to 3.6-3.7 cP in highly metastatic lines, consistent with greater macromolecular crowding and cytoplasmic reorganization reported in aggressive cells. Together, these results establish GEM-DDM as a fast, reproducible, and accessible platform for intracellular microrheology, providing new opportunities to link the physical state of the cytoplasm to cell function and disease progression. Statement of significancePhysical properties such as cytoplasmic viscosity influence how molecules move and interact within cells, affecting metabolism, signalling, and disease progression. Measuring viscosity in living cells has been technically challenging and often invasive. Here, we introduce GEM-DDM as a quantitative, non-invasive image-based analysis method combining genetically encoded multimeric nanoparticles (GEMs) with differential dynamic microscopy (DDM) to measure intracellular viscosity using standard wide-field microscopy. We validate its accuracy against established single particle tracking (SPT) methods and demonstrate its biological relevance by showing that cytoplasmic viscosity increases with metastatic potential in cancer cells. This approach provides an accessible platform for studying how the physical state of cells influences their function and pathology.

biophysics↗

bioRxiv2025

Beyond One-Size-Fits-All: Tumor Biology Influences Nanoparticle Behaviour in Cancer Models

Nanoparticles (NPs) are a promising tool for cancer therapy, yet few have successfully reached clinical application. Current nanomedicine development pipelines are focused on optimizing physical properties of NPs, overlooking the impact of tumor biology on their behavior. Here, we show that the same NPs exhibit distinct accumulation and penetration patterns in 3D spheroids derived from four tumor models (representative of lung, colon, breast, and cervical cancer). We uncover an inverse relationship between NP uptake and penetration: tumors with slower internalization show deeper NP diffusion. Proteomic analysis revealed that tumor-specific expression of endocytic and extracellular matrix proteins underlies this variability. Our findings challenge the prevailing one-size-fits-all approach and highlight the need to integrate tumor biology into NP design. Tailoring NPs to the unique cellular and extracellular features of each tumor type will be critical for developing more effective and clinically relevant nanotherapies.

cell biology↗