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 2015Slide 1
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 2016Slide 2
Hypothesis
The amount of cationic DOTAP, charge ratio and siRNA release can be varied to develop efficient and safe nanocarriers.
Introduction
Danielle Dennehy12 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
Danielle Dennehy
Current Treatments
NSAIDs -Ibuprofen
DMARDs -Methotrexate
Glucocorticosteroids -Prednisone
Biologics - Actemra
12 February 2016Slide 4
Danielle Dennehy
TNF-α
Pro-inflammatory cytokine
Pleiotropic
Produced by monocytes and macrophages
Can cause a positive feedback effect
12 February 2016Slide 5
Nature Reviews. Strand et al. 2007
Slide 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
Danielle Dennehy
Methods Part I: Nanoparticle Preparation
12 February 2016Slide 7
CH2Cl2 siRNA
PLGADOTAP
Evaporation by N2
90s 50W 60s 50W
Primary Emulsion
Secondary Emulsion
5ml2%PVA
Stirred 40min
1ml2%PVA
Sonication
Zetasizer
Results I
12 February 2016Slide 8
0
100
200
300
Size of 15% w/w DOTAP-PLGA Nanoparticles
N/P Ratios
nm
0.0
0.2
0.4
0.6
0.8
PDI of 15% w/w DOTAP-PLGA Nanoparticles
N/P Ratios
0
20
40
60
Zetapotential of 15% w/w DOTAP-PLGA Nanoparticles
N/P Ratios
mV
Empty 5 10 20 400
200
400
600
800
1000
Size of 15% w/w DOTAP-PLGA Nanoparticles
N/P Ratios
nm
Danielle Dennehy
Methods Part II: siRNA Extraction
Danielle Dennehy
12 February 2016Slide 9
CHCl3
HD SolutionNPs
WhirlmixerCentrifuge
siRNA Sample
TE Buffer
siRNA
Ribogreen
Encapsulation Efficiency
Danielle Dennehy12 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 1002003004005006000
10000200003000040000500006000070000
StandardLinear (Standard)Linear (Standard)N/P10N/P20NP30
ng
Fluor
esce
nce
0 100 200 300 400 500 6000
2000400060008000
100001200014000160001800020000
StandardLinear (Standard)N/P 10N/P 20
ng
Fluor
esce
nce
0 1 2 3 4 5 6 70
5000
10000
15000
20000
25000
30000
StandardLinear (Standard)Linear (Standard)Linear (Standard)NegC
ng
Fluor
esce
nce
0 2 4 6 8 10 12 140
5000100001500020000250003000035000
StandardLinear (Standard)Linear (Standard)NegC
ng
Fluor
esce
nce
Summary
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Slide 11
New drug delivery vehicle
Emerging therapy
Attractive therapeutic
Future Directions
Danielle Dennehy12 February 2016
Slide 12
In vitro TNF-α siRNA Release
Cell Transfection and Harvesting
Real-Time PCR
Toxicity Testing
Acknowledgements
Jana Hasse
Xianghui ZengCamilla Foged
In association with:siRNA based treatment of Rheumatoid ArthritisVaccine Design and DeliverySection for Biologics
Danielle Dennehy12 February 2016
Slide 13
Thank You!Tak!
Danielle Dennehy
Questions??
12 February 2016Slide 14
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