No significant effect between diluents was observed with respect to zeta potential. Figure 1 also illustrates the effect of N/P ratio on the size of chitosan-miRNA nanoparticles, with mean diameters of between 480 nm and 590 nm for N/P ratios of 50:1, 100:1, how to order 150:1, and 200:1. The inclusion of a crosslinking agent, TPP, in the manufacturing process significantly reduced the miRNA nanoparticles to as low as 115 �� 1.7 nm in diameter. Figure 1 Mean size (nm) of premiR-126-poly(ethyleneimine) (PEI) complexes prepared using phosphate-buffered saline (PBS) and 5% glucose, or premiR-126 chitosan complexes and premiR-126 chitosan-TPP nanoparticles. The percentage reduction in nanoparticle size using … The zeta potential of free miRNA was found to be ?15.98 �� 3.9 mV.

PEI-miRNA nanoparticles were positively charged above an N/P ratio of 5, while chitosan-miRNA nanoparticles had a net positive charge at all N/P ratios over 50:1 (data not shown). miRNA nanomedicine uptake into CFBE41o- cells: high content analysis High content analysis allows for analysis and quantification of multiple parameters of both cellular uptake of nanoparticles and cytotoxicity induced as a result of treatment. The IN Cell Analyzer 1,000 algorithm detects the presence of whole fixed cells by the presence of both the nucleus (stained with Hoechst 33342) and the cytoplasm (stained with F-actin using phalloidin FITC). Harnessing high content analysis, PEI-based nanoparticles at N/P ratios of 5:1 and 10:1 appear to be more effective at delivery of miRNA to the cell than chitosan-based or chitosan-TPP-based nanoparticles, or transfection using the commercially available RNA transfection agent, RiboJuice (Figure 2).

Figure 2B clearly indicates differences in the nature of the intracellular distribution of the miRNA within CFBE41o- cells when delivered using different carriers. Highly defined punctae are seen in PEI-miRNA-treated cells, while those treated with chitosan-miRNA nanoparticles show a diffuse distribution of miRNA in the cell. When TPP is used to prepare smaller and more defined miRNA nanoparticles, this can be seen to impact clearly the distribution of miRNA, with more defined areas of miRNA within the cell. These distribution differences can have a very significant effect on molecular kinetics and ultimately on the efficacy of the miRNA nanoparticles.

Figure 2 High content analysis of miRNA-Dy547 loaded nanoparticles association with CFBE41o- Cilengitide cells at 20.5 hours post transfection. (A) Comparative quantification of fluorescent miRNA delivered to CFBE cells by PEI- and chitosan-based nanoparticles at different … High content analysis of nanoparticle toxicity Cell count is the most obvious marker of toxicity. miRNA-PEI-based and miRNA-chitosan-based nanoparticles induced little or no toxicity at the N/P ratios tested (Figure 3).