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2. Vapor nanobubble is the more reliable photothermal mechanism for inducing endosomal escape of siRNA without disturbing cell homeostasis

Author

Fraire, Juan C., Houthaeve, Gaëlle, Liu, Jing, Raes, Laurens, Vermeulen, Lotte, Stremersch, Stephan, Brans, Toon, García-Díaz Barriga, Gerardo, De Keulenaer, Sarah, Van Nieuwerburgh, Filip, De Rycke, Riet, Vandesompele, Jo, Mestdagh, Pieter, Raemdonck, Koen, De Vos, Winnok H., De Smedt, Stefaan, and Braeckmans, Kevin

Published
2020
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8. Cas9 RNP transfection by vapor nanobubble photoporation for ex vivo cell engineering

Author

Raes, Laurens, Pille, Melissa, Harizaj, Aranit, Goetgeluk, Glenn, van Hoeck, Jelter, Stremersch, Stephan, Fraire, Juan C, Brans, Toon, de Jong, Olivier G, Maas - Bakker, Roel, Mastrobattista, Enrico, Vader, Pieter, de Smedt, Stefaan C, Vandekerckhove, Bart, Raemdonck, Koen, Braeckmans, Kevin, Afd Pharmaceutics, and Pharmaceutics

Subjects
Cell type, intracellular delivery, T cells, RM1-950, photoporation, MESENCHYMAL STEM-CELLS, Genome engineering, ACTIVATION, Cell therapy, DELIVERY, stem cells, Drug Discovery, Medicine and Health Sciences, Primary cell, gene editing, Chemistry, Mesenchymal stem cell, Biology and Life Sciences, Transfection, CAR, Cell biology, T-CELLS, Molecular Medicine, Therapeutics. Pharmacology, CRISPR-Cas9, Stem cell, SYSTEM, Intracellular
Abstract

The CRISPR-Cas9 technology represents a powerful tool for genome engineering in eukaryotic cells, advancing both fundamental research and therapeutic strategies. Despite the enormous potential of the technology, efficient and direct intracellular delivery of Cas9 ribonucleoprotein (RNP) complexes in target cells poses a significant hurdle, especially in refractive primary cells. In the present work, vapor nanobubble (VNB) photoporation was explored for Cas9 RNP transfection in a variety of cell types. Proof of concept was first demonstrated in H1299-EGFP cells, before proceeding to hard-to-transfect stem cells and T cells. Gene knock-out levels over 80% and up to 60% were obtained for H1299 cells and mesenchymal stem cells, respectively. In these cell types, the unique possibility of VNB photoporation to knock out genes according to user-defined spatial patterns was demonstrated as well. Next, effective targeting of the programmed cell death 1 receptor and Wiskott-Aldrich syndrome gene in primary human T cells was demonstrated, reaching gene knock-out levels of 25% and 34%, respectively. With a throughput of >200,000 T cells per second, VNB photoporation is a scalable and versatile intracellular delivery method that holds great promise for CRISPR-Cas9-mediated ex vivo engineering of cell therapy products.

Published
2021

13. Bubble‐Forming Films: Bubble Forming Films for Spatial Selective Cell Killing (Adv. Mater. 27/2021)

Author

Hua, Dawei, primary, Harizaj, Aranit, additional, Wels, Mike, additional, Brans, Toon, additional, Stremersch, Stephan, additional, De Keersmaecker, Herlinde, additional, Bolea‐Fernandez, Eduardo, additional, Vanhaecke, Frank, additional, Roels, Dimitri, additional, Braeckmans, Kevin, additional, Xiong, Ranhua, additional, Huang, Chaobo, additional, De Smedt, Stefaan C., additional, and Sauvage, Félix, additional

Published
2021
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14. Gasdermin E-dependent and -independent Subroutines Regulate the Passage of Dextrans During Apoptosis-driven Secondary Necrosis.

Author

De Schutter, Elke, primary, Ramon, Jana, additional, Pfeuty, Benjamin, additional, De Tender, Caroline, additional, Stremersch, Stephan, additional, Raemdonck, Koen, additional, de Beeck, Ken Op, additional, Declercq, Wim, additional, Riquet, Franck B., additional, Braeckmans, Kevin, additional, and Vandenabeele, Peter, additional

Published
2021
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15. Cas9 RNP transfection by vapor nanobubble photoporation for ex vivo cell engineering

Author

Afd Pharmaceutics, Pharmaceutics, Raes, Laurens, Pille, Melissa, Harizaj, Aranit, Goetgeluk, Glenn, van Hoeck, Jelter, Stremersch, Stephan, Fraire, Juan C, Brans, Toon, de Jong, Olivier G, Maas - Bakker, Roel, Mastrobattista, Enrico, Vader, Pieter, de Smedt, Stefaan C, Vandekerckhove, Bart, Raemdonck, Koen, Braeckmans, Kevin, Afd Pharmaceutics, Pharmaceutics, Raes, Laurens, Pille, Melissa, Harizaj, Aranit, Goetgeluk, Glenn, van Hoeck, Jelter, Stremersch, Stephan, Fraire, Juan C, Brans, Toon, de Jong, Olivier G, Maas - Bakker, Roel, Mastrobattista, Enrico, Vader, Pieter, de Smedt, Stefaan C, Vandekerckhove, Bart, Raemdonck, Koen, and Braeckmans, Kevin

Published
2021

17. Photoporation with Biodegradable Polydopamine Nanosensitizers Enables Safe and Efficient Delivery of mRNA in Human T Cells

Author

Harizaj, Aranit, primary, Wels, Mike, additional, Raes, Laurens, additional, Stremersch, Stephan, additional, Goetgeluk, Glenn, additional, Brans, Toon, additional, Vandekerckhove, Bart, additional, Sauvage, Félix, additional, De Smedt, Stefaan C., additional, Lentacker, Ine, additional, and Braeckmans, Kevin, additional

Published
2021
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18. Nanoparticle-sensitized photoporation enables inflammasome activation studies in targeted single cells

Author

Harizaj, Aranit, primary, Van Hauwermeiren, Filip, additional, Stremersch, Stephan, additional, De Rycke, Riet, additional, De Keersmaecker, Herlinde, additional, Brans, Toon, additional, Fraire, Juan C., additional, Grauwen, Karolien, additional, De Smedt, Stefaan C., additional, Lentacker, Ine, additional, Lamkanfi, Mohamed, additional, and Braeckmans, Kevin, additional

Published
2021
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19. Cytosolic delivery of gadolinium via photoporation enables improved in vivo magnetic resonance imaging of cancer cells

Author

Harizaj, Aranit, primary, Descamps, Benedicte, additional, Mangodt, Christophe, additional, Stremersch, Stephan, additional, Stoppa, Arianna, additional, Balcaen, Lieve, additional, Brans, Toon, additional, De Rooster, Hilde, additional, Devriendt, Nausikaa, additional, Fraire, Juan C., additional, Bolea-Fernandez, Eduardo, additional, De Wever, Olivier, additional, Willaert, Wouter, additional, Vanhaecke, Frank, additional, Stevens, Christian V., additional, De Smedt, Stefaan C., additional, Roman, Bart, additional, Vanhove, Christian, additional, Lentacker, Ine, additional, and Braeckmans, Kevin, additional

Published
2021
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21. Bubble Forming Films for Spatial Selective Cell Killing.

Author

Hua, Dawei, Harizaj, Aranit, Wels, Mike, Brans, Toon, Stremersch, Stephan, De Keersmaecker, Herlinde, Bolea‐Fernandez, Eduardo, Vanhaecke, Frank, Roels, Dimitri, Braeckmans, Kevin, Xiong, Ranhua, Huang, Chaobo, De Smedt, Stefaan C., and Sauvage, Félix

Published
2021
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22. Improved Label-Free Identification of Individual Exosome-like Vesicles with Au@Ag Nanoparticles as SERS Substrate

Author

Fraire, Juan C., primary, Stremersch, Stephan, additional, Bouckaert, Davinia, additional, Monteyne, Tinne, additional, De Beer, Thomas, additional, Wuytens, Pieter, additional, De Rycke, Riet, additional, Skirtach, Andre G., additional, Raemdonck, Koen, additional, De Smedt, Stefaan, additional, and Braeckmans, Kevin, additional

Published
2019
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23. Delivery of Mixed-Lineage Kinase Domain-Like Protein by Vapor Nanobubble Photoporation Induces Necroptotic-Like Cell Death in Tumor Cells

Author

Van Hoecke, Lien, primary, Raes, Laurens, additional, Stremersch, Stephan, additional, Brans, Toon, additional, Fraire, Juan C., additional, Roelandt, Ria, additional, Declercq, Wim, additional, Vandenabeele, Peter, additional, Raemdonck, Koen, additional, Braeckmans, Kevin, additional, and Saelens, Xavier, additional

Published
2019
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24. Gold Nanoparticle-Mediated Photoporation Enables Delivery of Macromolecules over a Wide Range of Molecular Weights in Human CD4+ T Cells

Author

Raes, Laurens, primary, Van Hecke, Clarissa, additional, Michiels, Julie, additional, Stremersch, Stephan, additional, Fraire, Juan C., additional, Brans, Toon, additional, Xiong, Ranhua, additional, De Smedt, Stefaan, additional, Vandekerckhove, Linos, additional, Raemdonck, Koen, additional, and Braeckmans, Kevin, additional

Published
2019
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26. Targeted Perturbation of Nuclear Envelope Integrity with Vapor Nanobubble-Mediated Photoporation

Author

Houthaeve, Gaëlle, primary, Xiong, Ranhua, additional, Robijns, Joke, additional, Luyckx, Bert, additional, Beulque, Yasmine, additional, Brans, Toon, additional, Campsteijn, Coen, additional, Samal, Sangram K., additional, Stremersch, Stephan, additional, De Smedt, Stefaan C., additional, Braeckmans, Kevin, additional, and De Vos, Winnok H., additional

Published
2018
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27. Exploring extracellular vesicles for siRNA delivery and Raman-based diagnostics

Author

Stremersch, Stephan, De Smedt, Stefaan, Raemdonck, Koen, and Braeckmans, Kevin

Subjects
Biology and Life Sciences
Abstract

During the past decade, extracellular vesicles (EVs) have been identified as important mediators of intercellular communication, enabling the functional transfer of bioactive molecules from one cell to another. Consequently, it is becoming increasingly clear that these vesicles are involved in many (patho)physiological processes, providing opportunities for therapeutic application. Moreover, it is known that the molecular composition of EVs reflects the physiological status of the producing cell and tissue, rationalizing their exploitation as biomarker for various diseases. In this dissertation EVs are explored as a new pharmaceutical tool, more specifically as a carrier for the delivery of RNAi-based therapeutics. In this respect, different isolation and cargo loading approaches are developed and evaluated. Additionally, the EV's ability to overcome the different cellular barriers is compared with current synthetic nucleic acid carriers. Besides their potential as a drug delivery vehicle, EVs also form an interesting biomarker source as they can be regarded as easy accessible, i.e. via liquid biopsy, windows on else difficult to reach cells. In this context a new platform to characterize individual EVs by surface-enhanced Raman spectroscopy is developed, which is able to discriminate between EVs from different cellular origin and has the potential to be further developed in a diagnostic context.

Published
2016

28. Cytosolic delivery of gadolinium viaphotoporation enables improved in vivomagnetic resonance imaging of cancer cellsElectronic supplementary information (ESI) available. See DOI: 10.1039/d1bm00479d

Author

Harizaj, Aranit, Descamps, Benedicte, Mangodt, Christophe, Stremersch, Stephan, Stoppa, Arianna, Balcaen, Lieve, Brans, Toon, De Rooster, Hilde, Devriendt, Nausikaa, Fraire, Juan C., Bolea-Fernandez, Eduardo, De Wever, Olivier, Willaert, Wouter, Vanhaecke, Frank, Stevens, Christian V., De Smedt, Stefaan C., Roman, Bart, Vanhove, Christian, Lentacker, Ine, and Braeckmans, Kevin

Abstract

Longitudinal in vivomonitoring of transplanted cells is crucial to perform cancer research or to assess the treatment outcome of cell-based therapies. While several bio-imaging techniques can be used, magnetic resonance imaging (MRI) clearly stands out in terms of high spatial resolution and excellent soft-tissue contrast. However, MRI suffers from low sensitivity, requiring cells to be labeled with high concentrations of contrast agents. An interesting option is to label cells with clinically approved gadolinium chelates which generate a hyperintense MR signal. However, spontaneous uptake of the label viapinocytosis results in its endosomal sequestration, leading to quenching of the T1-weighted relaxation. To avoid this quenching effect, delivery of gadolinium chelates directly into the cytosol viaelectroporation or hypotonic cell swelling have been proposed. However, these methods are also accompanied by several drawbacks such as a high cytotoxicity, and changes in gene expression and phenotype. Here, we demonstrate that nanoparticle-sensitized laser induced photoporation forms an attractive alternative to efficiently deliver the contrast agent gadobutrol into the cytosol of both HeLa and SK-OV-3 IP1 cells. After intracellular delivery by photoporation the quenching effect is clearly avoided, leading to a strong increase in the hyperintense T1-weighted MR signal. Moreover, when compared to nucleofection as a state-of-the-art electroporation platform, photoporation has much less impact on cell viability, which is extremely important for reliable cell tracking studies. Additional experiments confirm that photoporation does not induce any change in the long-term viability or the migratory capacity of the cells. Finally, we show that gadolinium ‘labeled’ SK-OV-3 IP1 cells can be imaged in vivoby MRI with high soft-tissue contrast and spatial resolution, revealing indications of potential tumor invasion or angiogenesis.

Published
2021
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29. Membrane vesicle secretion and prophage induction in multidrug-resistantStenotrophomonas maltophiliain response to ciprofloxacin stress

Author

Devos, Simon, primary, Van Putte, Wouter, additional, Vitse, Jolien, additional, Van Driessche, Gonzalez, additional, Stremersch, Stephan, additional, Van Den Broek, Wim, additional, Raemdonck, Koen, additional, Braeckmans, Kevin, additional, Stahlberg, Henning, additional, Kudryashev, Misha, additional, Savvides, Savvas N., additional, and Devreese, Bart, additional

Published
2017
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30. Identification of a novel mechanism of blood–brain communication during peripheral inflammation via choroid plexus‐derived extracellular vesicles

Author

Balusu, Sriram, primary, Van Wonterghem, Elien, additional, De Rycke, Riet, additional, Raemdonck, Koen, additional, Stremersch, Stephan, additional, Gevaert, Kris, additional, Brkic, Marjana, additional, Demeestere, Delphine, additional, Vanhooren, Valerie, additional, Hendrix, An, additional, Libert, Claude, additional, and Vandenbroucke, Roosmarijn E, additional

Published
2016
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34. Effect of Native Gastric Mucus on in vivo Hybridization Therapies Directed at Helicobacter pylori

Author

Santos, Rita S, primary, Dakwar, George R, additional, Xiong, Ranhua, additional, Forier, Katrien, additional, Remaut, Katrien, additional, Stremersch, Stephan, additional, Guimarães, Nuno, additional, Fontenete, Sílvia, additional, Wengel, Jesper, additional, Leite, Marina, additional, Figueiredo, Céu, additional, De Smedt, Stefaan C, additional, Braeckmans, Kevin, additional, and Azevedo, Nuno F, additional

Published
2015
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35. On-chip light sheet illumination enables diagnostic size and concentration measurements of membrane vesicles in biofluids

Author

Deschout, Hendrik, Raemdonck, Koen, Stremersch, Stephan, Maoddi, Pietro, Mernier, Guillaume, Renaud, Philippe, Jiguet, Sébastien, Hendrix, An, Bracke, Marc, Van Den Broecke, Rudy, Röding, Magnus, Rudemo, Mats, Demeester, Jo, De Smedt, Stefaan C., Strubbe, Filip, Neyts, Kristiaan, Braeckmans, Kevin, Deschout, Hendrik, Raemdonck, Koen, Stremersch, Stephan, Maoddi, Pietro, Mernier, Guillaume, Renaud, Philippe, Jiguet, Sébastien, Hendrix, An, Bracke, Marc, Van Den Broecke, Rudy, Röding, Magnus, Rudemo, Mats, Demeester, Jo, De Smedt, Stefaan C., Strubbe, Filip, Neyts, Kristiaan, and Braeckmans, Kevin

Abstract

Cell-derived membrane vesicles that are released in biofluids, like blood or saliva, are emerging as potential non-invasive biomarkers for diseases, such as cancer. Techniques capable of measuring the size and concentration of membrane vesicles directly in biofluids are urgently needed. Fluorescence single particle tracking microscopy has the potential of doing exactly that by labelling the membrane vesicles with a fluorescent label and analysing their Brownian motion in the biofluid. However, an unbound dye in the biofluid can cause high background intensity that strongly biases the fluorescence single particle tracking size and concentration measurements. While such background intensity can be avoided with light sheet illumination, current set-ups require specialty sample holders that are not compatible with high-throughput diagnostics. Here, a microfluidic chip with integrated light sheet illumination is reported, and accurate fluorescence single particle tracking size and concentration measurements of membrane vesicles in cell culture medium and in interstitial fluid collected from primary human breast tumours are demonstrated. © 2013 The Royal Society of Chemistry.

Published
2014

36. On-chip light sheet illumination enables diagnostic size and concentration measurements of membrane vesicles in biofluids

Author

Deschout, Hendrik, primary, Raemdonck, Koen, additional, Stremersch, Stephan, additional, Maoddi, Pietro, additional, Mernier, Guillaume, additional, Renaud, Philippe, additional, Jiguet, Sébastien, additional, Hendrix, An, additional, Bracke, Marc, additional, Van den Broecke, Rudy, additional, Röding, Magnus, additional, Rudemo, Mats, additional, Demeester, Jo, additional, De Smedt, Stefaan C., additional, Strubbe, Filip, additional, Neyts, Kristiaan, additional, and Braeckmans, Kevin, additional

Published
2014
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37. Intra- and Interspecies Effects of Outer Membrane Vesicles from Stenotrophomonas maltophiliaon β-Lactam Resistance

Author

Devos, Simon, Stremersch, Stephan, Raemdonck, Koen, Braeckmans, Kevin, and Devreese, Bart

Abstract

ABSTRACTThe treatment of Stenotrophomonas maltophiliainfection with β-lactam antibiotics leads to increased release of outer membrane vesicles (OMVs), which are packed with two chromosomally encoded β-lactamases. Here, we show that these β-lactamase–packed OMVs are capable of establishing extracellular β-lactam degradation. We also show that they dramatically increase the apparent MICs of imipenem and ticarcillin for the cohabituating species Pseudomonas aeruginosaand Burkholderia cenocepacia.

Published
2016
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38. Membrane vesicle secretion and prophage induction in multidrug-resistant Stenotrophomonas maltophilia in response to ciprofloxacin stress

Author

Devos, Simon, Van Putte, Wouter, Vitse, Jolien, Van Driessche, Gonzalez, Stremersch, Stephan, Van Den Broek, Wim, Raemdonck, Koen, Braeckmans, Kevin, Stahlberg, Henning, Kudryashev, Misha, Savvides, Savvas N., and Devreese, Bart

Subjects
biochemical phenomena, metabolism, and nutrition, bacterial infections and mycoses
Abstract

Several bacterial species produce membrane vesicles (MVs) in response to antibiotic stress. However, the biogenesis and role of MVs in bacterial antibiotic resistance mechanisms have remained unclear. Here, we studied the effect of the fluoroquinolone ciprofloxacin on MV secretion by Stenotrophomonas maltophilia using a combination of electron microscopy and proteomic approaches. We found that in addition to the classical outer membrane vesicles (OMV), ciprofloxacin-stimulated cultures produced larger vesicles containing both outer and inner membranes termed outer-inner membrane vesicles (OIMV), and that such MVs are enriched with cytosolic proteins. Remarkably, OIMV were found to be decorated with filamentous structures identified as fimbriae. In addition, ciprofloxacin stress leads to the release of bacteriophages and phage tail-like particles. Prophage induction by ciprofloxacin has been linked to pathogenesis and horizontal gene transfer in several bacterial species. Together, our findings show that ciprofloxacin treatment of S. maltophilia leads to the secretion of a heterogeneous pool of MVs and the induction of prophages that are potentially involved in adverse side-effects during antibiotic treatment.

39. Cas9 RNP transfection by vapor nanobubble photoporation for ex vivo cell engineering.

Author

Raes L, Pille M, Harizaj A, Goetgeluk G, Van Hoeck J, Stremersch S, Fraire JC, Brans T, de Jong OG, Maas-Bakker R, Mastrobattista E, Vader P, De Smedt SC, Vandekerckhove B, Raemdonck K, and Braeckmans K

Abstract

The CRISPR-Cas9 technology represents a powerful tool for genome engineering in eukaryotic cells, advancing both fundamental research and therapeutic strategies. Despite the enormous potential of the technology, efficient and direct intracellular delivery of Cas9 ribonucleoprotein (RNP) complexes in target cells poses a significant hurdle, especially in refractive primary cells. In the present work, vapor nanobubble (VNB) photoporation was explored for Cas9 RNP transfection in a variety of cell types. Proof of concept was first demonstrated in H1299-EGFP cells, before proceeding to hard-to-transfect stem cells and T cells. Gene knock-out levels over 80% and up to 60% were obtained for H1299 cells and mesenchymal stem cells, respectively. In these cell types, the unique possibility of VNB photoporation to knock out genes according to user-defined spatial patterns was demonstrated as well. Next, effective targeting of the programmed cell death 1 receptor and Wiskott-Aldrich syndrome gene in primary human T cells was demonstrated, reaching gene knock-out levels of 25% and 34%, respectively. With a throughput of >200,000 T cells per second, VNB photoporation is a scalable and versatile intracellular delivery method that holds great promise for CRISPR-Cas9-mediated ex vivo engineering of cell therapy products., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Published
2021
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40. Effect of Native Gastric Mucus on in vivo Hybridization Therapies Directed at Helicobacter pylori.

Author

Santos RS, Dakwar GR, Xiong R, Forier K, Remaut K, Stremersch S, Guimarães N, Fontenete S, Wengel J, Leite M, Figueiredo C, De Smedt SC, Braeckmans K, and Azevedo NF

Abstract

Helicobacter pylori infects more than 50% of the worldwide population. It is mostly found deep in the gastric mucus lining of the stomach, being a major cause of peptic ulcers and gastric adenocarcinoma. To face the increasing resistance of H. pylori to antibiotics, antimicrobial nucleic acid mimics are a promising alternative. In particular, locked nucleic acids (LNA)/2'-OMethyl RNA (2'OMe) have shown to specifically target H. pylori, as evidenced by in situ hybridization. The success of in vivo hybridization depends on the ability of these nucleic acids to penetrate the major physical barriers-the highly viscoelastic gastric mucus and the bacterial cell envelope. We found that LNA/2'OMe is capable of diffusing rapidly through native, undiluted, gastric mucus isolated from porcine stomachs, without degradation. Moreover, although LNA/2'OMe hybridization was still successful without permeabilization and fixation of the bacteria, which is normally part of in vitro studies, the ability of LNA/2'OMe to efficiently hybridize with H. pylori was hampered by the presence of mucus. Future research should focus on developing nanocarriers that shield LNA/2'OMe from components in the gastric mucus, while remaining capable of diffusing through the mucus and delivering these nucleic acid mimics directly into the bacteria.

Published
2015
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