Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.766254
Title: Manipulation and imaging of interactions between layer-by-layer capsules and live cells using nanopipettes and SICM
Author: Chen, Yuxiu
ISNI:       0000 0004 7654 0986
Awarding Body: Queen Mary University of London
Current Institution: Queen Mary, University of London
Date of Award: 2018
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Abstract:
Usability of many chemical substances with significant potential for biomedical applications is limited by their poor solubility in water or limited stability in the physiological environment. One of promising strategies for therapeutic targeted delivery of these types of substances into cells and tissues is their encapsulation inside polyelectrolyte microcapsules (Volodkin et al., 2004b, Sukhorukov et al., 1998b). Successful internalisation of microcapsules loaded with various macromolecules have been observed in several types of living cells (Javier et al., 2008, Kastl et al., 2013), however the mechanisms of the uptake of capsules by living cells are not yet fully understood. Detailed understanding of physico-chemical and mechanical interactions between capsules and living cells is required for specific targeting, effective delivery, and elimination of any potential toxic side effects. This has been largely limited by capabilities of available imaging techniques and lack of specific fluorescent markers for certain types of cellular uptake. The rate of internalisation of microcapsules was primarily studied at the level of cell population using conventional optical/fluorescence microscopy, confocal microscopy, and flow cytometry (Gao et al., 2016, Ai et al., 2005, Sun et al., 2015). These conventional fluorescence methods are known to be prone to overestimating the number of internalised capsules due to their limited capability to exclude capsules which were not fully internalised and remained attached to the cell surface (Javier et al., 2006). Experimental evidence with resolution high enough to resolve the fine membrane processes interacting with microcapsules has been limited to fixed samples imaged by scanning electron microscopy and transmission electron microscopy (Kastl et al., 2013) capturing randomly timed "snapshots" of what is likely to be highly dynamic and complex interaction. Physical force interactions between cellular membrane and capsules during the internalisation were suggested to cause buckling of capsules based on indirect evidence obtained using fluorescence microscopy in live cells 15 (Palankar et al., 2013) and separate measurements of capsule deformation under colloidal probe atomic force microscopy (AFM) outside of the cellular environment (Delcea et al., 2010, Dubreuil et al., 2003). However, our knowledge of the mechanical properties of the fine membrane structures directly involved in the internalisation process or how these structures form during the internalisation is very limited, if non-existent. Here we employ a different approach based on a high-resolution scanning probe technique called scanning ion conductance microscopy (SICM). SICM uses reduction in ionic current through the probe represented by an electrolyte-filled glass nanopipette immersed in saline solution to detect proximity of sample surface (Hansma et al., 1989, Korchev et al., 1997a). The technique has been previously used for high-resolution scanning of biological samples of complexity similar to what can be expected in case of microcapsules interacting with cells (Novak et al., 2014, Novak et al., 2009), and also for mapping mechanical properties at high resolution (Ossola et al., 2015, Rheinlaender and Schaffer, 2013). It has been proved to be able to visualise internalisation process of 200 nm carboxy-modified latex nanoparticles (Novak et al., 2014), however it is not clear whether it would be suitable for visualising internalisation of substantially larger, microscale-sized particles. The aim of this research was to visualize the live internalisation process of microcapsules entering cells by using SICM. The first two chapters of this thesis are introduction and literature review, which summarise the current state of the art. Chapter 3 states the aim and objectives of this study. Chapter 4 introduces the materials and methods we used in our research. Chapter 5, 6, 7 present the main findings of our research. Chapter 5 states the challenges we met in visualising the live internalisation of microcapsule as well as our solution for overcoming those challenges. At the end of that chapter, we describe the detailed procedure we used for recording the live internalisation of microcapsules. The results we got using this procedure are presented in chapter 6 and 7. In chapter 8, we discuss the results we found by comparing them to the results of previous research. In chapter 9, we summarise our study and give some suggestions on future work.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.766254  DOI: Not available
Keywords: Engineering and Materials Science ; multilayer microencapsulation technology ; Scanning Ion Conductance Microscopy
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