Silk has emerged as a promising contender for drug delivery applications, including the use of silk nanoparticles for anticancer drug delivery. Nanoparticles are internalised into cells via endocytosis; thus, silk nanoparticles have been proposed for lysosomotropic drug delivery. However, the intracellular fate of these nanoparticles has not been documented.One approach for quantifying the intracellular fate of silk nanoparticles is subcellular fractionation. Adequate, but gentle, homogenisation of cells, followed by characterisation of the subcellular fractions using biochemical markers, are key requirements for quantitative trafficking studies. Therefore, the aim of this thesis was to develop wet-lab tested biochemical marker assays for B16F10 cell subcellular fractions.The following biochemical marker assays were first scaled and refined: alkaline phosphatase to assay for the plasma membrane, succinate dehydrogenase for mitochondria, lactate dehydrogenase for the cytosol, N-acetyl-ß-glucosaminidase for lysosomes and DNA for the nucleus. A homogenisation scheme was then developed, using a cell cracker with 7 μm clearance. At four passes, this device provided over 80% ± 15 cell breakage, while retaining high lysosomal latency (82% ± 1 3.6), thereby providing a sample representative of the whole cell population.Trafficking studies require the use of non-toxic nanoparticle concentrations; therefore, silk nanoparticles were assessed for cytotoxicity in B16F10 cells using an MTT assay. The IC50 of silk nanoparticles was 17.58 μg/ml, using dextran (IC50 >200 μg/ml) and PEI (IC50 8.8 μg/ml) as negative and positive controls, respectively. These silk nanoparticles had a 131 nm diameter, with a polydispersity of 0.2, and a zeta potential in distilled water of -34.6 mV, as characterised by dynamic light scattering and zeta potential measurements, respectively.In conclusion, this thesis lays the groundwork for subsequent performance of wet-lab biomarker assays on subcellular fractions to quantify the intracellular trafficking of silk nanoparticles.
|Date of Award||1 Oct 2014|
- University Of Strathclyde
|Sponsors||EPSRC (Engineering and Physical Sciences Research Council)|
|Supervisor||Philipp Seib (Supervisor) & Clive Wilson (Supervisor)|