Real-time Label-free Multi-parametric Surface Plasmon Resonance Studies of Nanoparticle Uptake by Stem Cells
Ståhlberg, Roosa (2019)
Ståhlberg, Roosa
2019
Bioteknologian tutkinto-ohjelma
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2019-12-17
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-201911216171
https://urn.fi/URN:NBN:fi:tuni-201911216171
Tiivistelmä
Background and aims: Limbal stem cell deficiency (LSCD) is a group of corneal diseases, which can be very painful and cause blindness. LSCD is caused by dysfunctional or missing limbal epithelial stem cells (LESC), which take care of the corneal epithelium renewal. Novel tools to treat LSCD are needed, since bilateral LSCD-caused blindness cannot currently be treated. Light activatable polymeric nanoparticles could be applied in LESC in vitro genetical transfection before transplantation to patient’s eye. The used molecule in transfection e.g. small molecule, DNA or RNA could be loaded to nanoparticle and released with control of light. Aims of this thesis were to compare label-free nanoparticle uptake to labeled nanoparticle uptake with a real-time technique and to study LESC light activatable polymeric nanoparticle uptake, to find out which nanoparticle formulation would be the most prominent for cell transfection based on cellular uptake. Additionally, real-time measurement method was compared to previously used imaging-based measurement technique, which is possible to carry out only with fixed cells.
Methods: Real-time and label-free multi-parametric surface plasmon resonance (MP-SPR) technique was assessed to measure nanoparticle uptake by live LESCs and HeLa cells. HeLa cells were used for transferring method from one lab to another and to learn and validate the experimental setup, since HeLa cells have already been measured with MP-SPR. Three different light activatable polymeric nanoparticle formulations were studied (C11, P1C7 and P1C5). Both label-free nanoparticles and fluorescent-labeled nanoparticles were measured with MP-SPR. Additionally, confocal microscopy was employed to image labeled nanoparticle uptake in end-point analysis.
Results: HeLa cell method transfer with MP-SPR was successful. Protocol to measure LESCs was optimized. C11, the smallest nanoparticle sample, was the most efficiently uptaken by LESCs. Uptake was negatively affected when using labeled nanoparticles. MP-SPR and confocal microscopy results of cellular uptake were differed from each other and could not be compared.
Conclusions: Based on MP-SPR results, C11 is the most prominent nanoparticle for further in vitro studies to develop treatment and study LSCD. Real-time and label-free tools are needed, since labeling of these nanoparticles decreases significantly cellular uptake.
Methods: Real-time and label-free multi-parametric surface plasmon resonance (MP-SPR) technique was assessed to measure nanoparticle uptake by live LESCs and HeLa cells. HeLa cells were used for transferring method from one lab to another and to learn and validate the experimental setup, since HeLa cells have already been measured with MP-SPR. Three different light activatable polymeric nanoparticle formulations were studied (C11, P1C7 and P1C5). Both label-free nanoparticles and fluorescent-labeled nanoparticles were measured with MP-SPR. Additionally, confocal microscopy was employed to image labeled nanoparticle uptake in end-point analysis.
Results: HeLa cell method transfer with MP-SPR was successful. Protocol to measure LESCs was optimized. C11, the smallest nanoparticle sample, was the most efficiently uptaken by LESCs. Uptake was negatively affected when using labeled nanoparticles. MP-SPR and confocal microscopy results of cellular uptake were differed from each other and could not be compared.
Conclusions: Based on MP-SPR results, C11 is the most prominent nanoparticle for further in vitro studies to develop treatment and study LSCD. Real-time and label-free tools are needed, since labeling of these nanoparticles decreases significantly cellular uptake.