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Long-term live-cell microscopy with labeled nanobodies delivered by laser-induced photoporation

Nano Research 2020年第2期13卷 485-495页

作者： Jing Liu Tim Hebbrecht Toon Brans Eef Parthoens Saskia Lippens Chengnan Li Herlinde de Keersmaecker Winnok H.de Vos stefaan C.de smedt Rabah Boukherroub Jan Gettemans Ranhua Xiong Kevin Braeckmans Laboratory of General Biochemistry and Physical Pharmacy Faculty of PharmacyGhent UniversityGhent B-9000Belgium department of Biomolecular medicine Faculty of Medicine and Health SciencesGhent UniversityGhent B-9000Belgium VIB-UGent Center for Inflammation Research VIBGhent B-9000Belgium VIB Bioimaging Core Ghent VIBGhent B-9000Belgium department of Biomedical Molecular Biology Ghent UniversityGhent B-9000Belgium Univ.Lille CNRSCentrale LilleISENUniv.ValenciennesUMR 8520-IEMNLille F-59000Franc Laboratory of Cell Biology and Histology Department of Veterinary SciencesUniversity of Antwerp2020 AntwerpBelgium Centre for Advanced Light Microscopy Ghent UniversityGhent B-9000Belgium

Fluorescence microscopy is the method of choice for studying intracellular ***,its success depends on *** of specific and stable markers.A prominent example of markers that are rapidly gaining interest are nanobodies(Nbs.-15 kDa),which can be functionalized with bright and photostable organic *** to their relatively small size and high specificity,Nbs offer great potential for high-quality long-term subcellular imaging,but suffer from the fact that they cannot spontaneously cross the plasma membrane of live *** have recently discovered that laser-induced photoporation is well suited to deliver extrinsic labels to living cells without compromising their *** a laser-based technology,it is readily compatible with light microscopy and the typical cell recipients used for *** by these promising initial results,we demonstrate here for the first time successful long-term imaging of specific subcellular structures with labeled nanobodies in living *** illustrate this using Nbs that target GFP/YFP-protein constructs accessible in the cytoplasm,actin-bundling protein Fascin,and the histone H2A/H2B *** an efficiency of more than 80%labeled cells and minimal toxicity(-2%),photoporation proved to be an excellent intracellular delivery method for ***-lapse microscopy revealed that cell division rate and migration remained unaffected,confirming excellent cell viability and *** conclude that laser-induced photoporation labeled Nbs can be easily delivered into living cells,laying the foundation for further development of a broad range of Nbs with intracellular targets as a toolbox for long-term live-cell microscopy.

关键词： laser-induced photoporation vapor nanobubble long-term microscopy imaging nanobody intracellular delivery living cell labeling

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Intracellular delivery of mRNA in Adherent and Suspension Cells by Vapor Nanobubble Photoporation

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Nano-Micro Letters 2020年第12期12卷 404-420页

作者： Laurens Raes Stephan Stremersch Juan CFraire Toon Brans Glenn Goetgeluk Stijn de Munter Lien Van Hoecke Rein Verbeke Jelter Van Hoeck Ranhua Xiong Xavier Saelens Bart Vandekerckhove stefaan de smedt Koen Raemdonck Kevin Braeckmans Laboratory of General Biochemistry&Physical Pharmacy Ghent University9000 GhentBelgium Cancer Research Institute Ghent(CRIG) 9000 GhentBelgium department of Diagnostic Sciences Ghent University9000 GhentBelgium VIB-UGent Center for Medical Biotechnology 9052 GhentBelgium department of Biomedical Molecular Biology Ghent University9000 GhentBelgium department of Biochemistry and Microbiology Ghent University9000 GhentBelgium

Efficient and safe cell engineering by transfection of nucleic acids remains one of the long-standing hurdles for fundamental biomedical research and many new therapeutic applications,such as CAR T cell-based *** has recently gained increasing attention as a more safe and versatile alternative tool over viral-or DNA transposon-based approaches for the generation of adoptive T ***,limitations associated with existing nonviral mRNA delivery approaches hamper progress on genetic engineering of these hard-to-transfect immune *** this study,we demonstrate that gold nanoparticle-mediated vapor nanobubble(VNB)photoporation is a promising upcoming physical transfection method capable of delivering mRNA in both adherent and suspension *** transfection experiments on HeLa cells showed the importance of transfection buffer and cargo concentration,while the technology was furthermore shown to be effective for mRNA delivery in Jurkat T cells with transfection efficiencies up to 45%.Importantly,compared to electroporation,which is the reference technology for nonviral transfection of T cells,a fivefold increase in the number of transfected viable Jurkat T cells was ***,our results point toward the use of VNB photoporation as a more gentle and efficient technology for intracellular mRNA delivery in adherent and suspension cells,with promising potential for the future engineering of cells in therapeutic and fundamental research applications.

关键词： Transfection mRNA Photoporation Optoporation Gold nanoparticles Vapor nanobubbles

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聚合物纳/微纤维的制备及其应用

聚合物纳/微纤维的制备及其应用

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2014年全国高分子材料科学与工程研讨会

作者：黄超伯 Jo demeester stefaan C.de smedt 南京林业大学化学工程学院药学院根特大学(Ghent University)

＜正＞由静电纺丝技术制备的纳米单纤维和无纺布用途非常广泛,本文介绍静电纺丝纤维在生物医药方面的三种应用。1.防伪材料:由静电纺丝技术、激光切割、编码等技术制备的数字编码纤维（见图1A）具有以下新颖性:a.取代传统防伪技术,直接... 详细信息

＜正＞由静电纺丝技术制备的纳米单纤维和无纺布用途非常广泛,本文介绍静电纺丝纤维在生物医药方面的三种应用。1.防伪材料:由静电纺丝技术、激光切割、编码等技术制备的数字编码纤维（见图1A）具有以下新颖性:a.取代传统防伪技术,直接对药片本身而不是包装盒进行防伪;b.该材料存储信息量大、生物相容和成本不高,非常适合在不富裕的发展中国家推广。2.智能型材料在艾滋病防治中的应用:经过近30年的研究,疫苗的研制尚未成功

关键词： Electrospinning Memofibers HIV inhibition Cancer therapy

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Repeated photoporation with graphene quantum dots enables homogeneous labeling of live cells with extrinsic markers for fluorescence microscopy

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Light(Science & Applications) 2018年第1期7卷 583-592页

作者： Jing Liu Ranhua Xiong Toon Brans Saskia Lippens Eef Parthoens Francesca Cella Zanacchi Raffaella Magrassi Santosh K.Singh Sreekumar Kurungot Sabine Szunerits Hannelore Bové Marcel Ameloot Juan C.Fraire Eline Teirlinck Sangram Keshari Samal Riet de Rycke Gaëlle Houthaeve stefaan C.de smedt Rabah Boukherroub Kevin Braeckmans Laboratory of General Biochemistry and Physical Pharmacy Faculty of PharmacyGhent UniversityGhent B-9000Belgium Centre for Nano-and Biophotonics Ghent UniversityGhent B-9000Belgium VIB-UGent Centre for Inflammation Research VIBGhent B-9000Belgium VIB Bioimaging Core VIBGhent B-9000Belgium department of Biomedical Molecular Biology Ghent UniversityGhent B-9000Belgium Nanophysics(NAPH) Istituto Italiano di TecnologiaGenova 16163Italy Biophysics Institute(IBF) National Research Council(CNR)Via De Marini6-16149–GE GenovaItaly Physical and Materials Chemistry Division CSIR-National Chemical LaboratoryDr.Homi Bhabha RoadPune 411008India Academy of Scientific and Innovative Research Anusandhan Bhawan2 RafiMargNew Delhi 110001India Univ.Lille CNRSCentrale LilleISENUniv.ValenciennesUMR 8520-IEMNLille F-59000France Biomedical Research Institute Hasselt UniversityAgoralaan Building CDiepenbeek 3590Belgium Centre for Surface Chemistry and Catalysis KU LeuvenCelestijnenlaan 200FLeuven 3001Belgium Inflammation Research Center Image Core FacilityVIB9052 GhentBelgium department of Biomedical Molecular Biology Ghent University9052 GhentBelgium department of Plant Biotechnology and Bioinformatics Ghent University9052 GentBelgium Univ Lille 1 Univ Lille Nord FranceLab Phys Lasers Atomes&MolVilleneuve Dascq UMR 852359655France College of Chemical Engineering Jiangsu Key Lab of Biomass-based Green Fuels and ChemicalsNanjing Forestry University(NFU)Nanjing 210037China UMR 8523 Laboratoire de Physique des LasersAtomes et MoléculesUniversitéde LilleVilleneuve d’AscqFrance IEMN UMR 8520Universitéde LilleVilleneuve d’AscqFrance

In the replacement of genetic probes,there is increasing interest in labeling living cells with high-quality extrinsic labels,which avoid over-expression artifacts and are available in a wide spectral *** calls for a broadly applicable technology that can deliver such labels unambiguously to the cytosol of living ***,we demonstrate that nanoparticle-sensitized photoporation can be used to this end as an emerging intracellular delivery *** replace the traditionally used gold nanoparticles with graphene nanoparticles as photothermal sensitizers to permeabilize the cell membrane upon laser *** demonstrate that the enhanced thermal stability of graphene quantum dots allows the formation of multiple vapor nanobubbles upon irradiation with short laser pulses,allowing the delivery of a variety of extrinsic cell labels efficiently and homogeneously into live *** demonstrate high-quality time-lapse imaging with confocal,total internal reflection fluorescence(TIRF),and Airyscan superresolution *** the entire procedure is readily compatible with fluorescence(super resolution)microscopy,photoporation with graphene quantum dots has the potential to become the long-awaited generic platform for controlled intracellular delivery of fluorescent labels for live-cell imaging.

关键词： quantum enable replace

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同轴电纺聚氨酯/邻苯二甲酸醋酸纤维素pH值响应纤维应用于阴道内药物递送

同轴电纺聚氨酯/邻苯二甲酸醋酸纤维素pH值响应纤维应用于阴道内...

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2016年全国高分子材料科学与工程研讨会

作者：华大威刘中车王芳高步红陈飞张启路熊燃华韩景全 Sangram Keshari Samal stefaan C.de smedt 黄超伯化学工程学院江苏省生物质绿色能源与化学品重点实验室南京林业大学分析测试中心南京林业大学加拿大皇后大学(Queen's University)化学工程系高分子化学实验室赫尔辛基大学(University of Helsinki) 药学院根特大学(Ghent University) 材料科学与工程学院南京林业大学

＜正＞电纺纤维在诸多领域有广泛应用,其中包括生物医药,生物传感器,生物技术等[1]。本研究中我们采用同轴电纺技术制备了以聚氨酯为芯,以邻苯二甲酸醋酸纤维素为鞘的具有pH值响应的微纳纤维,并将其应用于阴道内药物递送。邻苯二甲酸醋...

＜正＞电纺纤维在诸多领域有广泛应用,其中包括生物医药,生物传感器,生物技术等[1]。本研究中我们采用同轴电纺技术制备了以聚氨酯为芯,以邻苯二甲酸醋酸纤维素为鞘的具有pH值响应的微纳纤维,并将其应用于阴道内药物递送。邻苯二甲酸醋酸纤维素（CAP）作为一种常用的药用辅料,其在pH值小于7时不溶解,而在pH大于7时溶解[2]。由于阴道内呈弱酸性,而人体精液pH为碱性,所以以CAP为鞘的同轴纤维可使其微纳纤维作为精液响应的药物递送

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