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Endocytic pathway of vascular cell adhesion molecule 1 in human umbilical vein endothelial cell identified in vitro by using functionalized nontoxic fluorescent quantum dots
KTH, School of Engineering Sciences (SCI), Applied Physics. (Hjalmar Brismar)ORCID iD: 0000-0002-2442-1809
KTH, School of Engineering Sciences (SCI), Applied Physics. 1989. (Ying Fu)ORCID iD: 0000-0002-3606-8985
RISE Acreo AB.
KTH, Centres, Science for Life Laboratory, SciLifeLab. KTH, School of Engineering Sciences (SCI), Applied Physics.ORCID iD: 0000-0003-0578-4003
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2019 (English)In: Sensors and actuators. B, Chemical, ISSN 0925-4005, E-ISSN 1873-3077, Vol. 297, article id 126702Article in journal (Refereed) Published
Abstract [en]

Studies about vascular cell adhesion molecule 1 (VCAM1) in tumor growth, metastasis, and angiogenesis suggest that targeting VCAM1 expression is an attractive strategy for diagnosis and anti-tumor therapy. However, the endocytic pathway of VCAM1 in vascular cells has not been well characterized. In this study we visualize the endocytic pathway of tumor necrosis factor α (TNFα) induced VCAM1 in human umbilical vein endothelial cell (HUVEC) in vitro using 5-carboxyfluorescein labeled VCAM1 binding peptides and fluorescent water-dispersible 3-mercaptopropionic acid (3MPA)-coated CdSe-CdS/Cd0.5Zn0.5S/ZnS core–multishell nontoxic quantum dots (3MPA-QDs) functionalized with VCAM1 binding peptides. Clear key in vitro observations are as follows: (a) 3MPA-QDs functionalized with VCAM1 binding peptides, denoted as VQDs, adhered and aggregated cumulatively to cell membrane around 2 h after VQD deposition to cell culture medium and were found in lysosomes in TNFα-treated HUVECs approximately 24 h after VQD deposition; (b) VQDs remained in TNFα-treated HUVECs for the whole 16 days of the experimental observation period; (c) quite differently, 3MPA-QDs were endocytosed then exocytosed by HUVECs via endosomes in about 24–48 h after 3MPA-QD deposition. Our study suggests that VCAM1 molecules, initially expressed on cell membrane induced by TNFα treatment, are internalized into lysosomes. This provides a novel means to deliver materials to lysosomes such as enzyme replacement therapy. Moreover, our meticulous sensing methodology of devising fluorescent nontoxic QDs advances biosensing technique for studying cellular activities in vitro and in vivo.

Place, publisher, year, edition, pages
Elsevier, 2019. Vol. 297, article id 126702
Keywords [en]
Bionanosensors, Colloidal fluorescent quantum dot, Endosome, Human umbilical vein endothelial cell, Lysosome, Vascular cell adhesion molecule 1 (VCAM1)
National Category
Industrial Biotechnology
Research subject
Physics, Biological and Biomedical Physics
Identifiers
URN: urn:nbn:se:kth:diva-262749DOI: 10.1016/j.snb.2019.126702ISI: 000478562700041Scopus ID: 2-s2.0-85067809943OAI: oai:DiVA.org:kth-262749DiVA, id: diva2:1362479
Note

QC 20191022

Available from: 2019-10-20 Created: 2019-10-20 Last updated: 2020-04-27Bibliographically approved
In thesis
1. Fluorescent quantum dots and graphene-based sensors for forensic applications
Open this publication in new window or tab >>Fluorescent quantum dots and graphene-based sensors for forensic applications
2019 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

A key emerging concept within the forensic sciences today areportable measurementdevices, where a much more efficient usage of the resources involved with crime-solving is possible if confirmatory measurements can be realised directly at a crimescene with such devices. Today, the majority of the presently used methods duringcriminal investigation at a crime scene involves measurements of a presumptivenature, which is a vital tool as it enables the screening of samples. In this thesis,the overarching goal is the development of tool kits for the analysis of biosampleson-site at a crime scene. This is mainly investigated through two routes: theusage of Quantum Dots (QDs) as a recognition element in sensory applications andfabrication of a graphene-based device for the detection of illicit drugs.The investigations conducted for the studies presented in this thesis focuses onsensory applications with a forensic detection scheme in mind: study I reveals in-trinsic properties of QDs to better understand sensing mechanisms upon bindinginteractions; study II demonstrates the fabrication of a graphene-based device forthe detection of illicit drugs; study III showcases the functionalised and bioconju-gated of QDs for a specific investigation into a biological process; study IV furtherthe investigation into the possible side-effects of QDs on biological specimens.In study I we numerically and experimentally investigate the intrinsic blinkingcharacteristics of CdSe-CdS/ZnS QDs. This includes a thorough examination of theexperimental parameters of the measurement setup: the bin time and excitationpower. Different mechanisms between the off- and on-state probability distributionsare found, wherein the on-state follows the random telegraph signal theory and theoff-state follows the inverse power law distribution.In study II, the detection of illicit drugs (amphetamine and cocaine) is achievedthrough graphene-based sensors processed to contain metal electrodes with superioradhesion and low contact resistance. The construction of a microfluidic system isfurther realised for a detection of molecules based on non-covalent interactions.With this system, a wavelength-dependent photoactivity for amphetamine and arange of its chemical analogs is demonstrated. A molecule dependent interactionwith the graphene surface is shown of the graphene surface either in the form ofp-doping (cocaine) or n-doping (amphetamine).Study III investigates the endocytic pathway of the vascular cell adhesionmolecule 1 (VCAM1) in Human Umbilical Vein Endothelial Cells (HUVECs) in-iiiivABSTRACTduced by Tumor Necrosis Factorα(TNFα) with the usage of 3-Mercaptopropionicacid coated (3MPA)-QDs and 5-Carboxyfluorescein (5FAM) functionalised and la-belled with VCAM1 binding peptides, respectively. Internalisation of the VCAM1molecules into lysosomes is shown with light microscopy through observations ofdifferent pathways of the 5FAM labelled peptides and functionalised QDs.In study IV we investigate the adverse effects of 3MPA-QDs on the humanairway epithelium by an examination of the calcium response in lung cells upon astimulation with QDs. The cellular response to the deposition of QDs is observedwith light microscopy and electrical measurements as a global increase of Ca2+in the epithelial layers and a transient decrease in the electrical response. Theseobservations imply that the influx of calcium caused by the QD deposition is inducedby mechanical stressIn an additional ongoing study, the age determination of dried blood spotsare investigated with the usage of protein markers commonly found in the blood.Human serum (HS) is spiked with a marker of interest to mimic those of normallevels in adult human males. After which the HS is allowed to undergo an ageingprocess in a 96 well plate and further analysed in terms of the enzymatic activitywith commercially available kits. The preliminary test results show that there is ameasurable change of activity dependenton the utilised marker that may act as abasis for the age determination of dried blod spots

Place, publisher, year, edition, pages
KTH Royal Institute of Technology, 2019. p. 119
Series
TRITA-SCI-FOU ; 2019:42
Keywords
Graphene, Quantum Dots, Blood, Forensics, Drugs
National Category
Natural Sciences Nano Technology
Research subject
Biological Physics
Identifiers
urn:nbn:se:kth:diva-262750 (URN)978-91-7873-307-1 (ISBN)
Presentation
2019-10-18, Pascal, Tomtebodavägen 23a, Solna, 10:00 (English)
Opponent
Supervisors
Funder
Swedish Foundation for Strategic Research , Forskningsinstitutsdoktorand 2015
Note

Examinator: Professor Björn Önfelt

Available from: 2019-10-21 Created: 2019-10-20 Last updated: 2020-02-18Bibliographically approved

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