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Biology Theses Department of Biology
8-3-2013
DesigningTargeted F4/80 Antibody Coated TNF-
α siRNA Loaded Nanoparticles: A Novel
Therapeutic Approach To Treat Inflammatory
Bowel Disease
Poonam Rakhya
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Rakhya, Poonam, "DesigningTargeted F4/80 Antibody Coated TNF-α siRNA Loaded Nanoparticles: A Novel Therapeutic Approach
To Treat Inflammatory Bowel Disease." Thesis, Georgia State University, 2013.
http://scholarworks.gsu.edu/biology_theses/48
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DESIGNING TARGETED F4/80 ANTIBODY COATED TNF-α siRNA LOADED
NANOPARTICLES: A NOVEL THERAPEUTIC APPROACH TO TREAT
INFLAMMATORY BOWEL DISEASE
by
POONAM RAKHYA
Under the Direction of Dr. Didier Merlin
ABSTRACT
Today, increasing evidences of systemic side effects with the treatments available for IBD
limit their therapeutic outcomes. The challenge is to target anti-TNF- agent to the inflamed
tissue. Here, we have demonstrated that TNF- siRNA can be efficiently loaded into F4-80
antibody (Ab) coated PLA-PEG-Mal nanoparticles (NPs) to target colonic macrophages
(MPs). The NPs were characterized for particle size, cytotoxicity, binding characteristics,
etc. These NPs are ~600 nm in diameter, non-cytotoxic, and are efficiently taken up by MPs
inhibiting TNF-α secretion by the MPs in vitro. In vivo, the release of TNF siRNA loaded
inside F4/80 Ab coated NPs into colitis mice model showed higher colitis attenuation (like
reduced weight loss, MPO activity and neutrophils infiltration) when compared to NPs not
covered. Thus, we have formulated an efficient targeted drug delivery system in which
TNF-α-siRNA NPs coated with F4/80 Ab represent an efficient therapeutic option for
diseases such as IBD.
INDEX WORDS: Nanoparticles, TNF- siRNA, Macrophages, IBD
DESIGNING TARGETED F4/80 ANTIBODY COATED TNF-α siRNA LOADED
NANOPARTICLES: A NOVEL THERAPEUTIC APPROACH TO TREAT
INFLAMMATORY BOWEL DISEASE
by
POONAM RAKHYA
A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of
Master of Science
in the College of Arts and Sciences
Georgia State University
2013
Copyright by
Poonam Rakhya
2013
DESIGNING TARGETED F4/80 ANTIBODY COATED TNF-α siRNA LOADED
NANOPARTICLES: A NOVEL THERAPEUTIC APPROACH TO TREAT
INFLAMMATORY BOWEL DISEASE
by
POONAM RAKHYA
Committee Chair: Dr. Didier Merlin
Committee: Dr. Andrew Gewirtz
Dr. Hamed Laroui
Electronic Version Approved:
Office of Graduate Studies
College of Arts and Sciences
Georgia State University
August 2013
iv
DEDICATION
This thesis is dedicated to my husband, Ashish,
for his endless love, support and encouragement.
v
ACKNOWLEDGEMENTS
With a deep sense of gratitude, I would like to express my sincere thanks to all who
were with me all the time and encouraged me during my work.
I owe a hearty debt of profound gratitude to my honorable mentor and advisor Dr.
Didier Merlin. I am indebted to him for suggesting the thesis topic to work on; his
systemic and constant supervision, inspiring guidance, encouragement and suggestions
propelled me into this journey and enabled me to accomplish this work. It has been my
proud privilege to work under such an esteemed, eminent and masterly guide.
I sincerely thank Dr. Hamed Laroui, Assistant Professor, for helping me in all
activities, for his elderly advice thinking about my betterment, and for constant
encouragement and good wishes he gave during my study period. It was a great privilege
for me to work with him. His kind attention, advice, towering support, incessant
encouragement, constant and vigilant supervision was a great esteem for me and I most
humbly express my thanks for his beneficent contributions.
I express my sincere gratitude towards my lab mates, Emilie Viennois, Moiz
Charania, Mark Baker, Saravanan Ayyadurai and Bo Xiao for their kind help & timely
advice that gave me the path to make it out very smoothly.
vi
TABLE OF CONTENTS
ACKNOWLEDGEMENTS.........................................................................................................v
LIST OF FIGURES.....................................................................................................................ix
LIST OF ABBREVIATIONS.....................................................................................................xi
1. INTRODUCTION.....................................................................................................................1
1.1 Epidemiology of IBD............................................................................................................1
1.2 Signs and symptoms of IBD.................................................................................................2
1.3 Treatments available for IBD..............................................................................................3
1.4 TNF-α and IBD....................................................................................................................4
1.5 RNA interference and IBD..................................................................................................5
1.6 Nanotechnology and IBD....................................................................................................7
1.7 Administration of nanoparticle based drugs in IBD...........................................................8
1.8 Goal of the proposed work...................................................................................................9
2. EXPERIMENTAL DETAILS...............................................................................................15
2.1 Synthesis of PLA-PEG-Maleimide and PLA-PEG-OH polymers....................................15
2.2 Preparation of TNF-α siRNA/PEI NPs covered with PVA..............................................15
2.3 Designing targeted F4/80 Ab coated TNF-α siRNA- loaded NPs....................................16
2.3.1 Synthesis of Fab’ portion of F4/80 Ab coated NPs......................................................16
2.3.2 Quantification of the number of Fab’F4/80 Ab fragments per nanoparticle...............17
2.4 Cell culture.........................................................................................................................18
2.5 Animal studies....................................................................................................................18
2.6 TNF-α siRNA/PEI polyplex formation and protection from PEI....................................18
vii
2.7 Particle size measurement..................................................................................................19
2.7.1 Photon correlation spectroscopy..................................................................................19
2.7.2 Scanning electron microscopy......................................................................................19
2.7.3 AFM Measurement.......................................................................................................19
2.8 Cellular Toxicity measurement..........................................................................................19
2.9 Interaction force measurement between F4/80 Ab coated NPs and MPs using
Surface Plasmon Resonance....................................................................................................20
2.10 Intracellular NP uptake visualization.............................................................................21
2.11 In vitro anti-inflammation effect of TNF-α siRNA-loaded NPs coated with
F4/80 Ab...................................................................................................................................22
2.12 In vivo anti-inflammation effect of TNF-α siRNA-loaded NPs coated with F4/80
Ab..............................................................................................................................................23
2.12.1 DSS induced colitis and oral administration of NPs..................................................23
2.12.2 Weight loss and Myeloperoxidase (MPO) activity measurement...............................23
2.12.3 Western Blotting.........................................................................................................24
2.12.4 Ly6G Staining and Counterstaining with Hematoxylin..............................................24
2.13 Flow cytometry analysis...................................................................................................25
2.14 Statistical Analysis............................................................................................................26
3. RESULTS................................................................................................................................29
3.1 Complexation/protection of TNF-α siRNA by PEI...........................................................29
3.2 Characterization of TNF-α siRNA-loaded NPs................................................................29
3.3 TNF-α siRNA-loaded NPs coated with F4/80 Ab do not affect cell viability and
may be used to safely transfect MPs........................................................................................30
viii
3.4 Nanoparticles coated with F4/80 Ab interact preferentially with RAW 264.7 cells
than Caco2-BBE cells..............................................................................................................30
3.5 Intracellular uptake of FITC-tagged siRNA by MPs is increased by using NPs
coated with Fab’ portion of F4/80 Ab.....................................................................................31
3.6 F4/80 Ab coated TNF siRNA-loaded NPs reduce TNF-α expression in inflamed
macrophages and shows in vitro anti-inflammation effect....................................................32
3.7 F4/80 Ab covered NPs loaded with TNF siRNA attenuate DSS-induced colitis
in mice and thus shows in vivo anti-inflammation effect.......................................................33
3.7.1 Weight loss and MPO activity analysis........................................................................33
3.7.2 Western Blotting analysis.............................................................................................34
3.7.3 Histology analysis.........................................................................................................34
3.8 Fab’ part of F4/80 Ab covered NPs loaded with TNF siRNA target intestinal
macrophages.............................................................................................................................35
4. DISCUSSIONS........................................................................................................................47
5. CONCLUSIONS.....................................................................................................................49
REFERENCES............................................................................................................................51
Description:TNF-α-siRNA NPs coated with F4/80 Ab represent an efficient therapeutic I owe a hearty debt of profound gratitude to my honorable mentor and
