investigating the drug loaded magnetic nanocomposite spheres for collagen induced arthritis ...

88 Pages·2014·5.4 MB·English

by  Ka Poh Man

INVESTIGATING THE DRUG LOADED MAGNETIC NANOCOMPOSITE SPHERES FOR COLLAGEN
INDUCED ARTHRITIS MURINE MODEL

A Thesis by
Ka Poh (Michelle), Man
Bachelor of Science, Wichita State University, 2007

Submitted to the Department of Mechanical Engineering
and the faculty of the Graduate School of
Wichita State University
in partial fulfillment of
the requirements for the degree of
Master of Science

July 2014
© Copyright 2014 by Man Ka Poh
All Rights Reserved
INVESTIGATING THE DRUG LOADED MAGNETIC NANOCOMPOSITE SPHERES FOR COLLAGEN
INDUCED ARTHRITIS MURINE MODEL

The following faculty members have examined the final copy of this thesis for form and content, and
recommend that it be accepted in partial fulfillment of the requirement for the degree of Master of Science
with a major in Mechanical Engineering.

_______________________________________
Ramazan Asmatulu, Committee Chair

_______________________________________
Paul H. Wooley, Committee Member

________________________________________
Ahmed Ikrammudin, Committee Member

iii
ACKNOWLEDGEMENTS

I would like to thank my adviser, Ramazan Asmatulu, for his guidance and being patient with
me during this process and his continual support. I would like to thank Elizabeth Clarkson for providing
statistical insight, Nora Strong and Zheng Song for helping and teaching me the processes and theory
portions of my research that pertains to Biological Sciences. Without their help, my research would not
have been successful. I would also like to extend my gratitude to members of my committee, Paul Wooley
and Ahmed Ikrammudin for their guidance and input on my research. Lastly I would like to thank my
friends for their moral support while I was working on this project.

iv
ABSTRACT

Rheumatoid arthritis is an incurable autoimmune disease that causes inflammation in the joints
that progresses and destroys its architecture. If left untreated this disease can spread to organs in the
body and loss of movement due to joint damage can result in a substantially reduced quality of life.
Treatment serves to alleviate symptoms and slow the progression of this disease but can cause a range of
side effects.
In this thesis a tested magnetic nanocomposite carrier is coupled with methotrexate, a proven
therapeutic agent for rheumatoid arthritis and used as a targeted drug delivery system to treat
experimental rheumatoid arthritis. A magnet was used as a targeting mechanism to draw drug carriers to
afflicted joints of collagen-induced arthritis in the murine model. Results show that use of the magnet
yielded statistical improvement compared to treatment without a targeting mechanism. Overall paw
scores and paw thicknesses showed lower measurements in mice treated with use of magnet than mice
treated without. The data showed a noticeable difference that can be stated with a 95% confidence
interval that use of targeted delivery systems prove effective.

v
TABLE OF CONTENTS

Chapter Page
1. INTRODUCTION ......................................................................................................................................... 1
2. LITERATURE REVIEW ............................................................................................................................... 3
2.1 Disease Pathology .......................................................................................................................... 3
2.2 Treatments ...................................................................................................................................... 4
2.3 Methotrexate (MTX) ...................................................................................................................... 6
2.4 Biomaterials .................................................................................................................................... 7
2.5 Magnetic Nanoparticles ................................................................................................................ 9

3. MATERIALS AND METHODS ................................................................................................................ 14
3.1 Animals ......................................................................................................................................... 14
3.2 Drug Delivery System ................................................................................................................. 14
3.3 Induction of Arthritis ................................................................................................................... 21
3.4 Treatment and Schedule ............................................................................................................. 22
3.5 MTT Assay for Determining DDS Release Rates ..................................................................... 23
3.6 Histology and Micro Computed Tomography ........................................................................ 24
3.7 Statistical Methods ....................................................................................................................... 28

4. RESULTS AND DISCUSSION .................................................................................................................. 30
4.1 Arthritic Development ................................................................................................................ 30
4.2 Treatment with DDS and Observations .................................................................................... 34
4.2.1 During Treatment .......................................................................................................... 35
4.3 Histology ....................................................................................................................................... 41
4.4 MCT Scan ...................................................................................................................................... 48
4.5 MTT Assay Results and DDS Imaging ...................................................................................... 52
4.6 Statistical data ............................................................................................................................... 56

5. CHALLENGES AND CONSIDERATIONS ............................................................................................ 58
6. CONCLUSION ........................................................................................................................................... 63
7. FUTURE STUDY ........................................................................................................................................ 64
REFERENCES ............................................................................................................................................................ 66

vi
TABLE OF CONTENTS (continued)

Chapter Page
APPENDICES ............................................................................................................................................................ 71
A. Sample Of Murine Arthritis Assesment Form ............................................................................. 72
B. Result Of Histopathology Scoring Based On Slide Review ....................................................... 73

vii
LIST OF TABLES

Table Page
1. Composition of DDS Synthesized ............................................................................................................ 16
2. Scoring Index used to Determine Progression of Disease in Mice ...................................................... 22
3. Scoring Used for Histopathology of Arthritis Observed in Mice Paws .............................................. 26
4. Table Indicating Dates of CIA Onset, Dosage, and Schedule of Treatment ....................................... 33
5. Absorbance Values For 96-Well Plate ...................................................................................................... 55

viii
LIST OF FIGURES

Figure Page
1. TEM image of nanocomposite. Scale indicates 400nm [31]. ................................................................. 11
2. Drug release rate of entrapped diclofenac sodium from nanoparticles with respect to incubation
time [31]. ...................................................................................................................................................... 11

3. Illustration of the morphology of magnetic nanocomposite sphere [34]. ........................................... 13
4. Vibra-Cell ultrasonic processor. ............................................................................................................... 17
5. Dispersant glass enveloped in Styrofoam with an overhead mixer in place. .................................... 17
6. Pulsing vortex mixer. ................................................................................................................................. 19
7. Set up of vacuum filtration system [36]. .................................................................................................. 20
8. Storage of fabricated drug carriers. .......................................................................................................... 21
9. Anatomy of front mouse paw [42]. .......................................................................................................... 27
10. Anatomy of the rear mouse paw [42]. ..................................................................................................... 27
11. Observed erythema and edema in (a) right front and (b) right rear paw ........................................... 30
12. Arthritis inflicted right rear paw. ............................................................................................................. 31
13. Mouse 580, treated – Visible swelling in paws. ...................................................................................... 32
14. Image shows the adhesion of a magnet to the base of the paw of a treated mouse. ......................... 34
15. Mice resting in cages after administration of treatment. ...................................................................... 35
16. Mouse 593 - Treated mice exhibit hair loss, indicated by arrows. ....................................................... 35
17. Plot of weight of mice during the period of treatment. Error bars represents standard error. ....... 36
18. Change in weight of the mice expressed in % weight lost or gained. ................................................. 38
19. Paw thickness of mice for the duration of treatment. Error bars represents standard error. .......... 39
20. Plot of the change in paw scores after initial onset of arthritis. ........................................................... 41
21. Treated mouse, HE staining, 100X – Left rear ankle of mouse 596. ..................................................... 42
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LIST OF FIGURES (continued)

Figure Page
22. Treated mouse, HE staining, 50X - Right front paw of mouse 581 (expired). .................................... 42
23. Treated mouse, HE staining, 40X - Right rear paw of mouse 591. ...................................................... 43
24. Treated mouse, HE staining, 100X - Image of right rear paw of mouse 591. ..................................... 44
25. Treated mouse, HE staining, 50X - Right front paw of mouse 580. ..................................................... 44
26. Treated mouse, HE Staining, 50X - Left rear paw of mouse 588. ......................................................... 45
27. Treated mouse, HE staining, 200X - Liver cells and DDS which are marked by the yellow
markers. ....................................................................................................................................................... 46

28. Spleen, 20X - (a) bright-field and (b) dark-field. .................................................................................... 47
29. Heart, 20X – (a) bright-field and (b) dark-field. ..................................................................................... 47
30. Liver, 20X – (a) bright-field, no dark particles observed and (b) dark-field. ...................................... 47
31. Paws of non-treated mouse, 583 – (a) left front paw, (b) left rear paw, (c) right rear paw. ............. 48
32. Treated, mouse 580 - (a) left rear paw and (b) right rear paw. Bone erosion is indicated by the
arrows. ......................................................................................................................................................... 49

33. Treated, mouse 588 – (a) right front paw and (b) left front paw. Bone erosion is indicated by the
arrows. ......................................................................................................................................................... 50

34. Treated, mouse 588 - (a) left rear paw (b) right rear paw. Bone erosion is indicated by the arrows.
....................................................................................................................................................................... 51

35. Non-treated, mouse 593 - (a) left rear paw and (b) right rear paw. Bone erosion is indicated by the
arrows. ......................................................................................................................................................... 51

36. Treated, mouse 590 - Right front paw. Bone erosion is indicated by the arrows. ............................. 52
37. Dark-field imaging of DDS at 100X. ........................................................................................................ 53
38. Dark-field imaging with green filter of dry DDS at 200X. .................................................................... 53
39. SEM images of DDS at 250X. .................................................................................................................... 54
40. Absorbance value plotted against concentration levels of DDS. ......................................................... 55
x