RNA interference (RNAi) is a naturally occurring pathway found in eukaryotes for gene silencing. Since the Nobel Prize winning
discovery of RNAi in 1998 scientists have been attempting to manipulate the pathway to selectively silence disease causing genes.
In theory, successful implementation of RNAi-based therapeutics will present the exciting prospect of silencing any diseasecausing
gene. The development of antisense therapeutics has been hampered by problems associated with delivery. A key
problem is that short interfering RNA (siRNA), the effector molecules in many therapeutic strategies, are incorporated into
acidic intracellular vesicles called endosomes and lysosomes. Moreover, inadequate tools for quantifying release from endosomes
and lysosomes are limiting the development of strategies aiming to improve the efficiency of intracellular trafficking. We have
used advances in nanotechnology to develop an analytical tool for following the path of formulations inside a cell by monitoring
changes in pH. We have used a micro-emulsion polymerisation technique to synthesise ratiometeric polyacrylamide based pHsensitive
biosensors, maximally fluorescent in endosomes and minimally fluorescent in the cytoplasm. Fluorescence microscopy
in conjunction with flow cytometry has shown positively charged biosensors internalise to endosomes and lysosomes of MRC-5
fibroblast cells. Quantitative analysis of fluorescence intensities has been used to make pH measurements, which can be used to
monitor endosomal release in a model system. The successful application of these biosensors will accelerate the development of
more effective siRNA formulations.
Arpan Desai is a currently a final year PhD student at the AstaZeneca Centre for Doctoral Training in Targeted Therpautics in the School of Pharmacy
at the University of Nottingham, UK. He received his BSc(hons) in Biotechnology from the University of Edinburgh. His research interest is in cellular
uptake and intracellular transport of siRNA formulations.
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