1. Potential for DOTAP-PLGA nanoparticles for the therapeutic
delivery of TNF-α siRNA in Rheumatoid Arthritis
BMOL40090: Research Project (Erasmus)
Presented by Danielle Dennehy
UNIVERSITY COLLEGE DUBLIN
12 February 2015
Slide 1

2. Aim
Investigate how different formulations based on DOTAP affects
the physicochemical properties, siRNA release, gene silencing
activity and cytotoxicity of DOTAP-PLGA nanocarriers loaded
with TNF-α siRNA for the use of a successful delivery vehicle in
the treatment of rheumatoid arthritis.
Danielle Dennehy
12 February 2016
Slide 2
Hypothesis
The amount of cationic DOTAP, charge ratio and siRNA release
can be varied to develop efficient and safe nanocarriers.

3. Introduction
Danielle Dennehy
12 February 2016
Slide 3
Unknown Cause
No Cure
Symptomatic Treatment
ProBoost Direct (2014). Available at: http://5296-presscdn-0-
7.pagely.netdna-cdn.com/wp-content/uploads/2013/08/RA-and-normal.jpg

4. Current Treatments
NSAIDs
-Ibuprofen
DMARDs
-Methotrexate
Glucocorticosteroids
-Prednisone
Biologics
- Actemra
Danielle Dennehy
12 February 2016
Slide 4

5. TNF-α
Pro-inflammatory cytokine
Pleiotropic
Produced by monocytes and macrophages
Can cause a positive feedback effect
Danielle Dennehy
12 February 2016
Slide 5
Nature Reviews. Strand et al. 2007

6. DOTAP-PLGA Nanoparticles
PLGA: Poly(DL-lactide-co-glycolide acid)
Copolymer of polylactic acid and polyglycolic acid
Biocompatible & Biodegradable
Non-condensing agent
DOTAP: Dioleoyltrimethylammoniumpropane
Cationic
Liposomal Transfection Reagent
High siRNA delivery efficiency
Danielle Dennehy
12 February 2016
Slide 6

7. Methods Part I: Nanoparticle Preparation
Danielle Dennehy
12 February 2016
Slide 7
CH2Cl2
siRNA
Evaporation
by N2
90s 50W 60s 50W
Primary
Emulsion
Secondary
Emulsion
5ml
2%PVA
Stirred 40min
1ml
2%PVA
Sonication
Zetasizer

8. Results I
12 February 2016
Slide 8
E
m
p
ty
5
1
0
2
0
0
1 0 0
2 0 0
3 0 0
S iz e o f 1 5 % w /w D O T A P -P L G A N a n o p a rtic le s
N /P R a tio s
nm
E
m
p
ty
5
1
0
2
0
4
0
0 .0
0 .2
0 .4
0 .6
0 .8
P D I o f 1 5 % w /w D O T A P -P L G A N a n o p a rtic le s
N /P R a tio s
E
m
p
ty
5
1
0
2
0
4
0
0
2 0
4 0
6 0
Z e ta p o te n tia l o f 1 5 % w /w D O T A P -P L G A N a n o p a rtic le s
N /P R a tio s
mV
E
m
p
ty 5
1
0
2
0
4
0
0
2 0 0
4 0 0
6 0 0
8 0 0
1 0 0 0
S iz e o f 1 5 % w /w D O T A P -P L G A N a n o p a rtic le s
N /P R a tio s
nm
Danielle Dennehy

9. Methods Part II: siRNA Extraction
Danielle Dennehy
12 February 2016
Slide 9
CHCl3
HD Solution
NPs
Whirlmixer
Centrifuge
siRNA
Sample
TE Buffer
siRNA
Ribogreen

10. Encapsulation Efficiency
Danielle Dennehy
12 February 2016
Slide 10
EE= 4.7%
EE:
N/P=10 = 20.9%
N/P=20 = 0.31%
EE:
N/P=10 = 48.56%
N/P=20 = 37.04%
N/P=30 = 225%
EE= 11.6%
0
10000
20000
30000
40000
50000
60000
70000
0 200 400 600
Fluorescence
ng
Standard
N/P10
N/P20
NP30
0
10000
20000
30000
40000
50000
60000
0 50 100 150
Fluorescence
ng
Standard
N/P 10
N/P 20
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
0 2 4 6
Fluorescence
ng
Standard
NegC
0
5000
10000
15000
20000
25000
30000
35000
0 5 10 15
Fluorescence
ng
Standard
NegC

11. Summary
Danielle Dennehy
12 February 2016
Slide 11
New drug delivery vehicle
Emerging therapy
Attractive therapeutic

12. Future Directions
Danielle Dennehy
12 February 2016
Slide 12
In vitro TNF-α siRNA Release
Cell Transfection and Harvesting
Real-Time PCR
Toxicity Testing

13. Acknowledgements
Jana Hasse
Xianghui Zeng
Camilla Foged
In association with:
siRNA based treatment of Rheumatoid Arthritis
Vaccine Design and Delivery
Section for Biologics
Danielle Dennehy
12 February 2016
Slide 13
Thank You!
Tak!

14. Questions??
Danielle Dennehy
12 February 2016
Slide 14