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KENYA JOSEPH
Interactions of siRNA Functionalized
Therapeutic RNA Nanoparticles with
Whole Blood & Isolated
Lymphocytes
• Introduction p. 3-5
• Research Objective p. 6
• What is siRNA? p. 7
• siRNAs as Nanoparticles p. 8
• siRNA Nanoparticle Production p. 9
• Process Advantages p. 10
• Methods p. 11
• Results p. 12-15
• Conclusions p. 16
• Discussion p. 17
• References p. 18
• Acknowledgments p. 19
• Questions p. 20
Table of Contents
www.home.ccr.cancer.gov
• Newest frontier of drug delivery and disease treatment
• Targets
• Cancers
• CNS and Brain Disorders
• Potential for infectious disease
• Why nucleic acids?
• fully customizable
• programmable
• can carry multiple functionalities
Introduction
• Nucleic acid based nanoparticles functionalized with
multiple short interference RNAs (siRNAs)
• Can be other therapeutic oligonucleotides
• Formulated with lipid-like carriers
• Can perform efficient intracellular delivery OR
• Can act as the active pharmaceutical ingredient itself
Introduction
• It is important to study how siRNA nanoparticles interact
with blood and lymphocytes
• to elucidate efficacy
• to examine blood stability
• Identify possible undesirable side-effects
Introduction
• 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.
• Observing three different siRNA nanoparticle designs, how well
do lymphocytes take up these particles?
• Are there differences in uptake between two different
transfection agents, Lipofectamine and PgP?
Research Objective
• Small or short interfering RNA
• Double-stranded RNA
• 20-25 base pairs
• Synthetic, micro RNA molecule
• Works within the RNA
interference (RNAi) pathway
• Interferes with expression of
specific genes with
complementary nucleotide
sequences by degrading mRNA
after transcription
What is siRNA?
www.scbt.com
siRNA as Nanoparticles
• Requirements for the nanoparticle carrier
• Protection of siRNA from degradation
• Enrichment of the siRNA in target tissue
• Facilitate cellular uptake of siRNA
• Why use siRNA?
• DNA is too large to diffuse across the cell membrane
• Effective targeting of specific tissue or cells
siRNA Nanoparticle Production
• Co-transcriptional One-Pot
Assembly
• Start with DNA templates for the
RNA nanoparticle of interest in a
transcription mixture
• Add T7 RNA Polymerase
• In vitro one-pot transcription
• RNA is formed and self-assembles
into functionalized RNA
nanoparticles
• The particles are purified
• The siRNAs are released by a cell’s
Dicer molecule
• Co-transcriptional One-Pot Assembly
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
Process Advantages
• 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 – 10 donors
• 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
50 ul:1 ml 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
Methods
• Figure 1 shows 2 fluorescent
shifts from the Whole Blood
Control
• Using Lipofectamine as
transfection agent
• shift 1 in red from the
introduction of the Nanoring
• shift 2 in blue and green from the
introduction of the Cube and
Duplex
• The Cube and Duplex experience
greater uptake with a larger shift
Results
• Figure 2 shows a fluorescent
shift from the Whole Blood
Control in Black
• Red - shift 1 from the Duplex
transfected with Lipofectamine
• Duplex transfected with PgP does
not show a shift (Blue)
• Pgp may not be effective with
duplex OR Pgp may be killing
cells
Results
• Figure 3 shows a fluorescent
shift from the Isolated
Lymphocytes control in Pink
• Blue - shift 1 from the Nanoring
transfected with Lipofectamine
Results
• Figure 4 shows a fluorescent
shift from the Isolated
Lymphocytes Control in Black
• Blue - shift 1 from the Duplex
transfected with Lipofectamine.
• Duplex transfected with PgP does
not show a shift (Red)
• Pgp may not be effective with
duplex OR Pgp may be killing
cells
Results
• 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
• Lymphocytes did not function as competently when isolated from whole
blood
• Construct design affects nanoparticle uptake but all 3 constructs
had uptake
• Nanoring & Cube design showed most effective uptake
• Lipofectamine worked better for transfection of these particles than
PgP – which may be killing cells
Conclusions
• The results 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 lymphocyte cells have the most efficient cellular
uptake by blood cells
• Ideal concentrations of nanoparticle administration in whole blood
• which lipid-like carrier works best for uptake
• mechanisms for cellular entry.
Discussion
This research was funded via internal funds from UNC
Charlotte. Thank you to Dr. K. Afonin and the
collaboration formed with Dr. J. Bennett of the
StressWAVES Biobehavioral Research Lab.
Acknowledgements
1. 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
References
Questions?

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Research - Oral Presentation

  • 1. KENYA JOSEPH Interactions of siRNA Functionalized Therapeutic RNA Nanoparticles with Whole Blood & Isolated Lymphocytes
  • 2. • Introduction p. 3-5 • Research Objective p. 6 • What is siRNA? p. 7 • siRNAs as Nanoparticles p. 8 • siRNA Nanoparticle Production p. 9 • Process Advantages p. 10 • Methods p. 11 • Results p. 12-15 • Conclusions p. 16 • Discussion p. 17 • References p. 18 • Acknowledgments p. 19 • Questions p. 20 Table of Contents www.home.ccr.cancer.gov
  • 3. • Newest frontier of drug delivery and disease treatment • Targets • Cancers • CNS and Brain Disorders • Potential for infectious disease • Why nucleic acids? • fully customizable • programmable • can carry multiple functionalities Introduction
  • 4. • Nucleic acid based nanoparticles functionalized with multiple short interference RNAs (siRNAs) • Can be other therapeutic oligonucleotides • Formulated with lipid-like carriers • Can perform efficient intracellular delivery OR • Can act as the active pharmaceutical ingredient itself Introduction
  • 5. • It is important to study how siRNA nanoparticles interact with blood and lymphocytes • to elucidate efficacy • to examine blood stability • Identify possible undesirable side-effects Introduction
  • 6. • 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. • Observing three different siRNA nanoparticle designs, how well do lymphocytes take up these particles? • Are there differences in uptake between two different transfection agents, Lipofectamine and PgP? Research Objective
  • 7. • Small or short interfering RNA • Double-stranded RNA • 20-25 base pairs • Synthetic, micro RNA molecule • Works within the RNA interference (RNAi) pathway • Interferes with expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription What is siRNA? www.scbt.com
  • 8. siRNA as Nanoparticles • Requirements for the nanoparticle carrier • Protection of siRNA from degradation • Enrichment of the siRNA in target tissue • Facilitate cellular uptake of siRNA • Why use siRNA? • DNA is too large to diffuse across the cell membrane • Effective targeting of specific tissue or cells
  • 9. siRNA Nanoparticle Production • Co-transcriptional One-Pot Assembly • Start with DNA templates for the RNA nanoparticle of interest in a transcription mixture • Add T7 RNA Polymerase • In vitro one-pot transcription • RNA is formed and self-assembles into functionalized RNA nanoparticles • The particles are purified • The siRNAs are released by a cell’s Dicer molecule
  • 10. • Co-transcriptional One-Pot Assembly 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 Process Advantages
  • 11. • 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 – 10 donors • 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 50 ul:1 ml 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 Methods
  • 12. • Figure 1 shows 2 fluorescent shifts from the Whole Blood Control • Using Lipofectamine as transfection agent • shift 1 in red from the introduction of the Nanoring • shift 2 in blue and green from the introduction of the Cube and Duplex • The Cube and Duplex experience greater uptake with a larger shift Results
  • 13. • Figure 2 shows a fluorescent shift from the Whole Blood Control in Black • Red - shift 1 from the Duplex transfected with Lipofectamine • Duplex transfected with PgP does not show a shift (Blue) • Pgp may not be effective with duplex OR Pgp may be killing cells Results
  • 14. • Figure 3 shows a fluorescent shift from the Isolated Lymphocytes control in Pink • Blue - shift 1 from the Nanoring transfected with Lipofectamine Results
  • 15. • Figure 4 shows a fluorescent shift from the Isolated Lymphocytes Control in Black • Blue - shift 1 from the Duplex transfected with Lipofectamine. • Duplex transfected with PgP does not show a shift (Red) • Pgp may not be effective with duplex OR Pgp may be killing cells Results
  • 16. • 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 • Lymphocytes did not function as competently when isolated from whole blood • Construct design affects nanoparticle uptake but all 3 constructs had uptake • Nanoring & Cube design showed most effective uptake • Lipofectamine worked better for transfection of these particles than PgP – which may be killing cells Conclusions
  • 17. • The results 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 lymphocyte cells have the most efficient cellular uptake by blood cells • Ideal concentrations of nanoparticle administration in whole blood • which lipid-like carrier works best for uptake • mechanisms for cellular entry. Discussion
  • 18. This research was funded via internal funds from UNC Charlotte. Thank you to Dr. K. Afonin and the collaboration formed with Dr. J. Bennett of the StressWAVES Biobehavioral Research Lab. Acknowledgements
  • 19. 1. 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 References