Biology subjects

Okkelman, I. A.

Publications and source records attributed to Okkelman, I. A..

4 recordsLinked to original sources

bioRxiv2026

Electrically Enhanced Intraperitoneal Drug Delivery Resolved by Fluorescence Lifetime Imaging Microscopy

Intraperitoneal delivery of nanoparticles (NPs) holds promise in the treatment of peritoneal metastases (PM), and the use of electric fields may enhance their tissue penetration. Evaluating NP delivery requires evidence of intracellular payload release, which is challenging in autofluorescent tissues. Here, we combined electromotive drug administration (EMDA) with cationic RL-100 NPs co-loaded with doxorubicin (DOX) and a near-infrared aza-BODIPY tracer, reporting carrier-associated distribution, while phasor fluorescence lifetime imaging microscopy (FLIM) characterized the molecular environment of DOX. In colorectal carcinoma, NP-delivered DOX progressed from cytoplasmic puncta to nuclear accumulation, while carrier-associated fluorescence remained cytoplasmic, supporting intracellular release. EMDA enhanced NP penetration in ex vivo human peritoneum and healthy rat peritoneum. In a syngeneic rat model of colorectal PM, both free and NP-delivered DOX remained detectable to approximately 1200 um after EMDA, compared with < 200 um after passive delivery. NP-delivered DOX remained detectable 24 h after EMDA. Whole-animal and ex vivo imaging showed the highest carrier-associated signals in tumor nodules and tumor-bearing omentum. These findings support electrically enhanced intraperitoneal delivery and a dual-reporter approach to evaluate carrier distribution, drug penetration, and intracellular release, with potential applications in other autofluorescent tissues. Together, these capabilities provide a preclinical basis for investigating NP-assisted delivery.

pharmacology and toxicology↗

bioRxiv2025

Fluorescence Lifetime Imaging Microscopy (FLIM) visualizes internalization and biological impact of nanoplastics in live intestinal organoids

The increasing micro- and nanoplastic (MNP) pollution poses significant risks to human and animal health, yet the mechanisms of their accumulation and effects on absorptive tissues such as the gastrointestinal tract remain poorly understood. Addressing these knowledge gaps requires tractable models coupled to dynamic live cell imaging methods, to enable multi-parameter analysis at single cell resolution. Here we report a new method combining adult stem cell-derived small intestinal organoid cultures with multi-parameter live Fluorescence Lifetime Imaging Microscopy (FLIM) to study MNP interactions with gut epithelium. To facilitate this, we optimized live imaging of porcine and mouse small intestinal organoids with an apical-out topology. Subsequently, we produced a set of pristine MNPs based on PMMA and PS (<200 nm, doped with deep-red fluorescent dye) exhibiting different surface charges, and evaluated their interaction with organoids displaying controlled epithelial polarity. We found that nanoparticles differently interacted with apical and basal membranes of the organoids and even showed a species-specific pattern of cellular uptake. Using a phasor-FLIM approach, we demonstrate better sensitivity of FLIM over conventional intensity-based microscopy. The fluorescence lifetime barcoding enabled distinguishing different types of MNP and their interaction sites within organoids. Finally, we studied short (1 day)- and long (3 days)-term exposure effects of PMMA and PS-based MNPs on mitochondrial function, total energy budget and epithelial inflammation and found that even pristine MNPs could disrupt chemokine production and mitochondrial membrane potential in intestinal epithelial cells. The presented FLIM approach will advance the study of MNP toxicity, their biological impacts on gastrointestinal tissue and help tracing other types of fluorescent nanoparticles in live organoid and 3D ex vivo systems.

pharmacology and toxicology↗

bioRxiv2023

Live microscopy of multicellular spheroids with the multi-modal near-infrared nanoparticles reveals differences in oxygenation gradients

Assessment of hypoxia, nutrients, metabolite gradients, and other hallmarks of the tumor microenvironment within 3D multicellular spheroid and organoid models represents a challenging analytical task. Here, we report red/near-infrared emitting cell staining O2-sensitive nanoparticles, which enable measurements of spheroid oxygenation on a conventional fluorescence microscope. Nanosensor probes, termed MMIR (multi-modal infrared), incorporate a near-infrared O2-sensitive metalloporphyrin (PtTPTBPF) and a deep red aza-BODIPY reference dyes within a biocompatible polymer shell, allowing oxygen gradients quantification via fluorescence ratio and phosphorescence lifetime readouts. We optimized staining techniques and evaluated nanosensor probe characteristics and cytotoxicity. Subsequently, we applied nanosensors to the live spheroid models based on HCT116, DPSCs, and SKOV3 cells, at rest and treated with drugs affecting cell respiration. We found that the growth medium viscosity, spheroids size, and formation method influenced spheroid oxygenation. Unexpectedly, some spheroids (produced from HCT116 and dental pulp stem cells) exhibited inverted oxygenation gradients, with higher core oxygen levels than the periphery. This contrasted with the frequently encountered normal gradient of hypoxia towards the core caused by diffusion. Further microscopy analysis of spheroids with an "inverted" gradient demonstrated metabolic stratification of cells within spheroids: thus, autofluorescence FLIM of NAD(P)H revealed the formation of glycolytic core, and localization of OxPhos-active cells at the periphery. Collectively, we demonstrate a strong potential of NIR-emitting ratiometric nanosensors for advanced microscopy studies targeting live and quantitative real-time monitoring of cell metabolism and hypoxia in complex 3D tissue models. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=38 SRC="FIGDIR/small/571110v2_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@49cef8org.highwire.dtl.DTLVardef@1b31072org.highwire.dtl.DTLVardef@fcaf4aorg.highwire.dtl.DTLVardef@1583b5f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2020

Extracellular Ca2+-sensitive fluorescent protein biosensor based on a collagen-binding domain

The importance of extracellular gradients of biomolecules becomes increasingly appreciated in the processes of tissue development and regeneration, in health and disease. In particular, dynamics of extracellular calcium concentration is rarely studied. Here, we present low affinity Ca2+ biosensor based on Twitch-2B fluorescent protein fused with the cellulose- and collagen-binding peptides. These recombinant chimeric proteins can bind cellulose and collagen scaffolds and enable for scaffold-based biosensing of Ca2+ in proximity of live 3D tissue models. We found that the Twitch-2B mutant is compatible with intensity-based ratiometric and fluorescence lifetime imaging microscopy (FLIM) measurement formats, under one- and two-photon excitation modes. Furthermore, the donor fluorescence lifetime of ColBD-Twitch displays response to [Ca2+] over a range of [~]2-2.5 ns, making it attractive biosensor for multiplexed FLIM microscopy assays. To evaluate performance of this biosensor in physiological measurements, we applied ColBD-Twitch to the live Lgr5-GFP mouse intestinal organoid culture and measured its responses to the changes in extracellular Ca2+ upon chelation with EGTA. When we combined it with spectrally resolved FLIM of lipid droplets using Nile Red dye, we observed changes in cytoplasmic and basal membrane-associated lipid droplet composition in response to the extracellular Ca2+ depletion, suggesting that intestinal epithelium can respond to and compensate such treatment. Altogether, our results demonstrate ColBD-Twitch as a prospective Ca2+ sensor for multiplexed FLIM analysis in a complex 3D tissue environment.

bioengineering↗