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