Difference between revisions of "Topic4-Differentially Charged Hollow Core/Shell Lipid–Polymer–Lipid Hybrid Nanoparticles for Small Interfering RNA Delivery"
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Revision as of 16:43, 23 November 2012
siRNA, double emulsion nanoparticles, lipids, vesicles
Small Interfering RNA (SiRNA) has the ability to silence genes that are responsible for causing diseases. However, the delivery of siRNA to cells is made difficult through the negative charge interactions the nucleotides of the siRNA have with the cell membrane. As a result, many research groups have looked into using nanocarriers to transport the siRNA through the cell membrane at which point the siRNA is released and can effectively suppress the expression of a targeted gene sequence. Shi et al explored the use of lipid based carriers that were formulated through a double emulsion method. The lipids formed a vesicle that entrapped the siRNA in the hydrophilic core. The surface of the lipid particle contained polyethylene glycol (PEG) chains which has been known to increase circulation in the blood stream and evade an immune system attack (the exact reason for this is not known). This unique particle has several different components: a hollow core formed by a positively charged lipid layer (the negative siRNA can be easily entrapped by the positive charge of the lipid), and a hydrophobic polymer, poly(lactic-co-glycolic acid) (PLGA), that connects the hydrophobic tails of the inner lipid layer to the outer lipid-PEG layer. The PLGA layer is used as a barrier to slow the release of siRNA from the particle.
These nanoparticles were formulated using a double emulsion technique. This technique involves the use of a sonicator probe that sends out high frequency sound waves instigating the self assembly of the independant elements of the system such as the PLGA polymer and two different types of lipid. The directionality of the lipid formation (hydrophobic centre vs. hydrophilic centre) is dependant on the ratio of organic solvent to aqueous solution used. Double emulsion particles are formed with two sonications. The first sonication is performed using a high ratio of organic phase to aqueous phase. The organic phase contains the hydrophobic PLGA polymer and lipid. The aqueous phase contains siRNA. A high ratio of an organic solvent to aqueous phase ensures that the first sonication forms particles with a hydrophilic core and the hydrophobic tails facing outward into the polymer. The second sonication involves the addition of an aqueous phase containing a different lipid (the lipid is below the critical micelle concentration in the water) in which the ratio of the organic solvent to the aqueous phase is low. This ensures that the hydrophilic heads of the lipid are facing outward and the particles are suspended in water. Figure 1 illustrates the final particle.
Dynamic light scattering was used to observe the size of the particles formed using this double emulsion technique. The particle size was found to be around 225 nm. The ability of the particle to sufficiently encapsulate the siRNA was also investigated. The encapsulation efficiency was found to be 78-82% which compared to other particles reported in literature is significantly higher. The release of various types of siRNA (GFP siRNA, Luciferase siRNA, and GAPDH siRNA) from the nanoparticle was observed over a period of 225 hours. The data demonstrated an initial burst release of siRNA from the particle in the first 10-20 hours in which 50% of the siRNA was released. Then the release slowed down to reach a plateau. The curve in Figure 2 demonstrates that sustained release of the therapeutic content of the particle can be achieved. The final experiment performed to evaluate therapeutic efficacy was the transfection of Green Fluorescent Protein (GFP) expressing HeLa cells with GFP silencing siRNA. The success of the nanoparticle would be determined by the ability of the nanoparticle (NP) encapsulating GFP siRNA to reduce the expression of the GFP in the cells. An industry standard called Lipofectamine (labelled as Lipo2000 in the plot) was used as a positive control. Lipofectamine is a lipid based carrier that can encapsulate siRNA and can also be used successfully in transfection experiments. The goal is to achieve as much silencing using the nanoparticle encapsulating siRNA as the lipofectamine. According to Figure 3, it is clear that a quantity of 30-60 pmol of siRNA can achieve as much of a silencing effect as Lipo2000.