RESULTS

Advantages• Programmability (different modules at different ratios)• High one-pot yields of assembly (>90%)• Relatively small sizes (Rh(nanocube)~6 nm, Rh(nanoring)~8nm)• Relatively short sequences (nanocube 52 nts, nanoring 44 nts)• Tunable thermodynamic and chemical stabilities• Ability to introduce multiple different functionalities• Can be formed co-transcriptionally

Isolated

Lymphocyte

Layer

Flow Cytometry Schematic

• Co-transcriptional assembly of functionalized siRNA nanoparticles with Alexa fluorescent tags using T7 polymerase

• Healthy adults were brought into the lab between 7:30 and 8:30a

• Whole Blood was drawn and Lymphocytes isolated from each sample using a Histopaque isolation assay

• siRNA nanoparticles were transfected using Lipofectamine and PgP and incubated for 30 minutes at room temperature

• Whole Blood and isolated Lymphocytes were plated and diluted twice at 50ul:1ml of PBS

• Transfected nanoparticles were introduced to the blood and lymphocyte samples, comparing samples with transfection added prior to or after dilution

• Samples were analyzed using BD Accuri C6 flow cytometer to observe fluorescent shift

Kenya Joseph1 and Kirill Afonin2

1Biology, 2Chemistry, The University of North Carolina at Charlotte

Interactions of siRNA Functionalized Therapeutic RNA Nanoparticles with Whole Blood and Isolated Lymphocytes

Figure 1 Figure 2 Figure 3 Figure 4

• Figure 1 shows 2 fluorescent shifts from the Whole Blood Control – shift 1 in red from the introduction of the Nanoring and shift 2 in blue and green from the introduction of the Cube and Duplex

• Figure 2 shows a fluorescent shift from the Whole Blood Control in black – shift 1 from the Duplex transfected with Lipofectamine. Duplex transfected with PgP does not show a shift

• Figure 3 shows a fluorescent shift from the Lymphocyte control in pink – shift 1 from the Nanoring transfected with Lipofectamine

• Figure 4 shows a fluorescent shift from the Lymphocyte Control in black – shift 1 from the Duplex transfected with Lipofectamine. Duplex transfected with PgP does not show a shift

• The newest frontier of drug delivery and disease treatment is nanotechnology utilizing therapeutic nucleic acids, which are proving to be fully customizable, programmable and can carry multiple functionalities.

• Nucleic acid based nanoparticles functionalized with multiple short interference RNAs (siRNAs), or other therapeutic oligonucleotides and formulated with lipid-like carriers for efficient intracellular delivery can act as an active pharmaceutical ingredient.

• It is important to study how siRNA nanoparticles interact with blood and lymphocytes to elucidate efficacy and possible undesirable side-effects.

CONCLUSIONS• Experimentation has thus far shown a marked difference

in nanoparticle uptake when the formulation is introduced after the samples are serially diluted as opposed to introduction to undiluted human blood samples.

• Construct design affects nanoparticle uptake. • This may indicate that the currently used experimental

protocol may affect apoptosis and cell morphology of lymphocytes thus promoting their interaction with nanoparticles.

• Further experimentation aims to examine which constructs and cells have the most efficient cellular uptake by blood cells, which lipid-like carrier works best for uptake and mechanisms for cellular entry.

METHOD

REFERENCES1. Afonin, K. A., Bindewald, E., Yaghoubian, A. J., Voss, N., Jacovetty, E., Shapiro, B. A., & Jaeger,

L. (2010). In vitro assembly of cubic RNA-based scaffolds designed in silico. Nature Nanotechnology, 5(9), 676–682. http://doi.org/10.1038/nnano.2010.160

2. Afonin, K. A., Grabow, W. W., Walker, F. M., Bindewald, E., Dobrovolskaia, M. A., Shapiro, B. A., & Jaeger, L. (2011). Design and self-assembly of siRNA-functionalized RNA nanoparticles for use in automated nanomedicine. Nature Protocols, 6(12), 2022–2034. http://doi.org/10.1038/nprot.2011.418

3. Afonin, K. A., Kireeva, M., Grabow, W. W., Kashlev, M., Jaeger, L., & Shapiro, B. A. (2012). Co-transcriptional Assembly of Chemically Modified RNA Nanoparticles Functionalized with siRNAs. Nano Letters, 12(10), 5192–5195. http://doi.org/10.1021/nl302302e

4. Afonin, K. A., Viard, M., Kagiampakis, I., Case, C. L., Dobrovolskaia, M. A., Hofmann, J., … Shapiro, B. A. (2015). Triggering of RNA Interference with RNA–RNA, RNA–DNA, and DNA–RNA Nanoparticles. ACS Nano, 9(1), 251–259. http://doi.org/10.1021/nn504508s

5. Grabow, W. W., Zakrevsky, P., Afonin, K. A., Chworos, A., Shapiro, B. A., & Jaeger, L. (2011). Self-Assembling RNA Nanorings Based on RNAI/II Inverse Kissing Complexes. Nano Letters, 11(2), 878–887. http://doi.org/10.1021/nl104271s

INTRODUCTION BACKGROUND RESULTS

CONCLUSIONS

REFERENCES

• To examine the cellular uptake by whole blood and by lymphocyte isolations from human donors of several fluorescently tagged functional RNA nanoparticles selected from the laboratory library.

OBJECTIVE

This research was funded via internal funds from UNC Charlotte. Thank you to Dr. Afonin and the collaboration formed with Dr. Bennett of the StressWAVES Biobehavioral Research Lab.

ACKNOWLEDGEMENTS

Yingling YG & Shapiro BA. Computational design of an RNA hexagonal nanoring and an RNA nanotube. Nano Lett. 7, 2007

Afonin KA, Bindewald E, Yaghoubian AJ, Voss N, Jacovetty E, Shapiro BA, Jaeger L. In vitro assembly of cubic RNA-based scaffolds designed in silico. Nature Nanotechnology, 5, (2010)

Programmable RNA-based nanoscaffolds

Grabow WW, Zarkrevsky P, Afonin KA, Chworos A, Shapiro BA, Jaeger L. Self-assembling RNA nanorings based on RNAI/II

inverse kissing complexes. Nano Letters. 11, 2011