Figure 3. NP-siRNA delivered using serum-free media, which prevents NP-siRNA aggregation, results in significantly greater uptake and pH-responsive membrane lysis.

Fluorescent siRNA was complexed to NPs at a charge ratio (+/−) = 4 and delivered to hMSCs in vitro in DMEM + 10% FBS or serum-free OptiMEM media and uptake was detected via flow cytometry. A) NP-siRNA delivered in serum-free OptiMEM media results in significantly increased median fluorescence intensity (MFI) 24 hours post-treatment compared to DMEM + 10% FBS at 30 nM siRNA. A 3-fold lower NP-siRNA dose (10 nM) delivered in OptiMEM achieves similar uptake levels as 30 nM NP-siRNA dose in DMEM + 10% FBS. n=6, **p<0.01, ****p<0.0001 compared to untreated, #p<0.0001. B) TEM micrograph reveals NP structures (arrow) with diameters similar to values obtained by DLS are visible in hMSC membrane bound vesicles 6 hours post-treatment in serum-free OptiMEM media in vitro, and were no longer detectable at later time points. C) TEM micrograph shows NP structures (arrow) with diameters consistent with aggregated NPs from DLS experiments that accumulate in intracellular vesicles 24 hours post-treatment when delivered in DMEM + 10% FBS. D) Hemolysis data shows pH-responsive membrane lysis activity is significantly lower in serum-mediated NP-siRNA aggregated state at endosomal pH 6.2 and 5.6. Representative data from a single experiment using four sample replicates, ***p<0.001; Significance determined using two-way ANOVA with Dunnett’s test to compare to no treatment (NT) control and Tukey’s test for multiple pairwise comparisons. Error bars represent standard deviation.