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Title: Formulation of non-ionic surfactant vesicles for therapeutic delivery of siRNA in cancer treatment
Author: Obeid, Mohammad Ali Radi
ISNI:       0000 0004 6422 6121
Awarding Body: University of Strathclyde
Current Institution: University of Strathclyde
Date of Award: 2017
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RNA interference (RNAi) is a post-transcriptional gene regulatory mechanism that involves the degradation of a target messenger RNA (mRNA) through the incorporation of short interfering RNAs (siRNA) which is complementary to the target mRNA. Unmodified, naked siRNA is unstable and cannot freely penetrate the cell membrane. The application of siRNA based therapeutics is limited by the development of an effective delivery system to deliver therapeutic siRNA to the cytoplasm of the target cells. Lipid-based nanoparticles, such as liposomes, are the most commonly investigated systems for siRNA delivery. However, another type of lipid-based system known as non-ionic surfactant vesicles (NISV) which are commonly used for drug delivery of various therapeutic agents, are relatively safe and non-expensive have not been extensively studied for siRNA delivery. Therefore, the aim of this study was to investigate the potential of NISV in siRNA delivery. Different manufacturing methods are used for the preparation of NISV and most of them are limited to bench scale and cannot be used on a larger industrial scale. This project sought to optimise the formulation method of NISV and to investigate their potential to effectively deliver siRNA to tumour cells in vitro and in vivo. Different methods of NISV manufacturing were compared including: thin-film hydration method (TFH), heating method, and microfluidic mixing. The formation of spherical nanoparticles was confirmed by examining the morphology of the NISV prepared by the three methods with atomic force microscopy (AFM) or scanning electron microscopy (SEM). TFH and heating methods were able to produce small (< 200 nm) and homogeneous NISV only after using a post-manufacturing size reduction step such as extrusion. This was time consuming and it was difficult to control batch to batch variations. Microfluidic mixing was found to produce NISV of the desired size and dispersity required for regulatory approval, in a single step, without the need of size reduction and homogenisation. Moreover, the preparation time was significantly reduced with controllable parameters, which suggested this method would make production feasible on a larger scale. Therefore, microfluidic mixing was chosen to prepare different NISV formulations and to investigate the optimisation of the factors related to this method, including the mixing time, mixing ratio, and the type of hydration media used. These were found to have significant effects on the physical characteristics of the vesicles such as size, polydispersity index, and charge. Particle size was shown to be decreased significantly (p < 0.05) by increasing the ratio between the aqueous and lipid phase as well as by increasing the total flow rates in the mixing process. Optimum ratios were found to be 3:1 between the aqueous and lipid phase at a total flow rates of 12 ml/min. Moreover, changing the type of aqueous media used to prepare the particles also resulted in significant effects on the particle size, dispersity, and charge. Smaller particles (desirable for siRNA delivery) were obtained using distilled water (DW), Tris, and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffers while the use of phosphate buffered saline (PBS) or normal saline (NS) resulted in the preparation of larger particles. Therefore, for the next experiments that involved siRNA transfections, NISV were prepared with DW. After optimising all these parameters, in vitro studies were conducted with the NISV formulations made with either monopalmitin glycerol (MPG) or Tween85 (T85) as a non-ionic surfactant, in addition to cholesterol and dimethyldioctadecylammonium bromide (DDAB) as a charging material, at different molar ratios of these components. Cytotoxicity evaluation of the prepared NISV formulations were carried out on non-small lung cancer cells (A549), human melanoma cancer cells (A375), breast cancer cells (A780), and mouse melanoma cells (B16-F10-LUC). These experiments were carried out with the use of normal human prostate cells PNT2) as a control. The used cancer cell lines were selected as they are among the most abundant cancers types worldwide. Cytotoxicity studies indicated that all the NISV formulations were not toxic at or below 40 μg/ml. The prepared NISV formulations had high siRNA encapsulation efficiency (~90%). Fluorescent microscope and flow cytometry studies on A549 cells, using fluorescent labelled negative control siRNA loaded in all the NISV formulations tested, indicated high cellular uptake by the cells. These uptake results were confirmed with B16-F10-LUC mouse melanoma cells, where the prepared NISV were able to successfully deliver siRNA into the cells compared to naked siRNA, which was not taken up by the cells. Following these experiments that proved cellular uptake of siRNA delivered by NISV, siRNA targeting either green fluorescent protein (GFP) in copGFP-A549 cells, or luciferase enzyme in B16-F10-LUC cells were encapsulated in NISV that contained either MPG or T85 as a non-ionic surfactant. Inhibition of GFP expression by anti-GFP siRNA (siGFP) delivered using different NISV formulations was evaluated by fluorescence measurement, flow cytometry, polymerase chain reaction, and Western blotting studies. These results indicated that all the NISV formulations were able to deliver siGFP to the cells and significantly (p<0.05) suppress GFP expression. These results were confirmed by transfecting the luciferase producing B16-F10-LUC cells with anti-luciferase siRNA (siLUC) using the same NISV formulations. Measuring the level of luciferase expression after siLUC transfections using a luciferase protein assay system successfully demonstrated the suppression of luciferase expression. Among all the NISV prepared, significant GFP and luciferase gene knockdown results were achieved when using NISV that contained T85 as the non-ionic surfactant. This superior formulation was then used in in vivo experiments using nude BALB/c mice inoculated with B16-F10-LUC cells that induce melanoma cancer-expressing luciferase. After intra-tumoural injection with this formulation, siLUC was delivered to the cells and suppressed luciferase expression at a significantly (p<0.05) higher level than mice treated with naked siLUC. These in vivo results confirm the ability of NISV to successfully delivery siRNA into the cytoplasm of the target tumour cells and suppress the target protein. In conclusion, NISV prepared by microfluidics have been demonstrated extensively and for the first time to have the potential to be used as a delivery system for siRNA. These results have shown that NISV can be used to overcome the barriers, such as low stability and poor cellular uptake, in siRNA-based therapeutics. NISV are a promising delivery system which can be investigated more extensively to target different over-expressed proteins in the process of developing different effective cancer medications.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral