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.
With the Undergraduate project reports now marked, Gemma has kindly agreed to post her project report on the blog. You can find a pdf of the report here.
As most of the methods and results are already on the blog, a slightly abridged and edited version of the discussion section is replicated below:
As discussed in a previous post, the punctate distribution of all of the SmartFlares (SFs) is both unexpected and curious. In order to better understand where the SFs are localised in the cell, we would like to do a fairly routine co-staining experiment.
The SmartFlare that we have chosen for this project is specific to vascular endothelial growth factor (VEGF) mRNA.
VEGF was selected as the target because it’s transcript becomes increased in a cell as part of a characterized response to hypoxia with the aid of hypoxia inducible factor-1 (HIF-1). Active HIF-1 is a heterodimer of α and β subunits, levels of HIF-1α are kept low by oxygen-dependent prolyl hydroxylases (PHDs). When a cell becomes hypoxic, PHD levels are reduced so more HIF-1α is present to dimerise with HIF-1β forming the active heterodimer that can act as the VEGF transcription factor.