The purpose of this work is to develop a carrier system for delivering RNA molecules aimed to downregulate specific functions in T cells. In many forms of cancer, T cells that express the protein Forkhead Box P3 (Foxp3) are associated with cancer progression. These cells can be identified by CD4 and CD25, molecules express on the cell surface. Studies have shown that downregulation of Foxp3 can increase the ability of other immune cells to destroy tumors. A class of RNA molecules, commonly referred to as “siRNA”, bind to and degrade specific messenger RNA (mRNA) in a sequence-dependent manner such that expression of the encoded protein is terminated. Because mRNA molecules are located inside cells, a carrier system is required to facilitate the uptake of siRNA, which does not passively diffuse through the plasma membrane. To this end, nanosized polymeric particles coated with the polycation, ornithinex10-histidinex6 (or O10H6) were used to adsorb siRNA that bind to the mRNA encoding Foxp3. The RNA-loaded particles are spherical and uniform in size (normally distributed, polydispersity index = 0.072). Loading of RNA to the particles was confirmed using gel electrophoresis. RNA complexed with the particles are protected from serum destabilization: 83.1% of RNA were recovered compared to 36.1% in RNA that were not associated with the particles. Association with the particles increased the uptake of the RNA in mouse T cells from 3.2±0.2% (free RNA) to 20.1±3.9%. Specifically, uptake of the RNA in T cells that express CD4 increased from 2.7±0.2% to 27.1±1.3% when particles were employed. These differences are statistically significant in three experiments conducted (p < 0.01). Internalization of the RNA into T cells was confirmed using confocal imaging. Flow cytometric analysis showed that the particle-complexed RNA reduced the percentage of T cells that express both CD4 and CD25 in mice carrying tumors from 24.0% when free RNA molecules were used to 13.5%. In these cells, the level of Foxp3 mRNA was reduced by 30%. In conclusion, the particles facilitate the uptake of siRNA molecules into a population of T cells that is known to promote cancer growth.
Phagocytic Uptake of Particles by Immune cells Under Flow Conditions