This week I have been evaluating osmotic lysis of pinocytic vesicles as a technique for cellular delivery. In the preliminary stages of my project I will be loading dextran into the cells.
This week I have been evaluating scrape-loading as a technique for cellular delivery. In the preliminary stages of my project I will be loading dextran into the cells.
My name is Ellie Madders and I am in my third year at the University of Liverpool studying Genetics. I am undertaking my Honours project supervised by Dr Raphael Levy.
The aim of my project is to deliver gold nanoparticles into the cytosol and demonstrate the targeting of organelles. To investigate this the nanoparticles will be conjugated with various peptides and several delivery methods will be evaluated.
First the techniques to deliver GNPs will be assessed. The chosen delivery methods are scrape-loading in which the culture medium is replaced with GNPs and the cells scraped with a rubber policeman to introduce the GNPs into the cell (McNeil, P. et.al. 1984) and osmotic lysis of pinocytic vesicles (Figure 1).
Once a superior method has been evaluated I will aim to target GNPs to the nucleus by conjugation to the TAT peptide, peptide sequence; WGRRVRRRIRRPPPPPPPPPP (Field et al., 2015). Further organelles that could be targeted could include localisation to the Endoplasmic reticulum via conjugation to the CALNNR8 peptide (Sun et al., 2008).
Field, L. D., Delehanty, J. B., Chen, Y. and Medintz, I. L. 2015. Peptides for specifically targeting nanoparticles to cellular organelles: Quo vadis ? Accounts of Chemical Research, 48 (5), 1380-1390.
McNeil, P. L., Murphy, R. F., Lanni, F. and Taylor, D. L. 1984. A Method for Incorporating Macromolecules into Adherent Cells,(4),1556.
Okada, C. Y. and Rechsteiner, M. 1982. Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles. Cell, 29 (1), 33-41.
Sun, L. L., Liu, D. J. and Wang, Z. X. 2008. Functional gold nanoparticle-peptide complexes as cell-targeting agents. Langmuir, 24 (18), 10293-10297.
Nuclear Localisation Signal (NLS) is a peptide sequence that tags certain proteins for nuclear transport. By conjugating NLS to gold nanoparticles (GNPs), this should (in theory) result in the GNPs being transported to the nucleus. This can then be detected using photothermal microscopy. However, actually putting this into practice is slightly harder. Due to the abundance of positively charged residues in the NLS sequence they can cause the nanoparticles to aggregate.
Following the preparation of 10nm GNPs conjugated to CALNN, the next step was to use these for microinjection and then photothermal imaging. For this I used 35mm gridded dishes, this meant that the coordinates of successfully injected cells could be recorded to make the cells easier to find again. The length of time the cells were removed from the incubator had to be kept to a minimum as after extended time periods they would begin to die. After the cells had been injected they were then fixed using paraformaldehyde and stored in the fridge until needed for imaging.
Gold nanoparticles (GNPs, also known as “colloidal gold“) have become the subject of large amounts of research in recent years which can mainly be attributed to their unique properties. Their potential applications range from targeted drug delivery to cell imaging. It is their use in cell imaging, specifically photothermal microscopy, that I’m interested in for my summer project.
Microinjection is one of the key techniques I will be using during my summer project. It is an incredibly versatile and widely used technique, with applications from ranging from IVF to producing transgenic organisms. Its application during my project will be to introduce the gold nanoparticles (GNPs) into the cells for photothermal imaging.