MAGNETIC AND ALBUMIN TARGETED DRUG DELIVERY FOR BREAST CANCER
TREATMENT
A Thesis by
Farhana Abedin
Bachelor of Science, Bangladesh University of Engr. & Tech., 2008
Submitted to the Department of Mechanical Engineering
and the faculty of the Graduate School
Wichita State University
in partial fulfillment of
the requirements for the degree of
Master of Science
July 2011
© Copyright 2011 by Farhana Abedin
All Rights Reserved
MAGNETIC AND ALBUMIN TARGETED DRUG DELIVERY FOR BREAST CANCER
TREATMENT
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
_________________________________
Shang-You Yang, Committee member
_________________________________
Hamid Lankarani, Committee member
iii
DEDICATION
To my parents and loving husband
iv
ACKNOWLEDGEMENTS
The author would like to express her heartiest gratitude to her supervisor, Dr. Ramazan
Asmatulu, Assistant professor, Department of Mechanical Engineering for his invaluable
guidance and support in accomplishing this research work. The author is very grateful to her
committee member Dr. Shang-You-Yang, Associate professor, Department of Biological
Sciences for his advice and suggestions. The author would also like to thank her committee
member Dr. Hamid Lankarani, Professor, Department of Mechanical Engineering for his
important comments.
The author is thankful to Zheng Song, Laboratory manager CIBOR and Nora Zacharias,
Animal lab manager CIBOR, for their support in all biological experiments. The author
expresses her deepest gratefulness to Dr. Kevin Langenwalter, NMR/Instrumentation manager,
Department of Chemistry for his guidance and assistance in HPLC tests. The author is deeply
thankful to Md. Rajib Anwar, graduate student, Department of Mechanical Engineering for
supporting and helping her in this research work.
The author is thankful to Heath Misak, graduate student, Department of Mechanical
Engineering and Janani Sri Gopu for their assistance in this research work.
Finally the author is very grateful to her parents, family members and her husband, Md.
Rajib Anwar for their inspiration and support to take this research work to completion.
v
ABSTRACT
This research work involves multifunctional magnetically targeted drug delivery
microspheres for treatment against breast cancer. A combination therapy approach was followed
by encapsulating two chemotherapeutics, 5-Fluorouracil (5-Fu) and cyclophosphamide in
poly(D, L-lactide-co-glycolide) (PLGA) microspheres. Magnetite nanoparticles and albumin
were also incorporated in the microspheres to achieve targeted treatment. The microspheres were
fabricated using oil-in-oil emulsion/solvent evaporation technique. Albumin is attracted to cancer
cells and thus it is likely to draw the microspheres towards tumor cells. On application of
magnetic field near tumor site, magnetites in the microspheres are likely to guide them to the
region of magnetic field. This will allow release of drugs from microspheres in the cancer cells.
Also the burst release of drugs and then slow release due to diffusion in the cancer cells lead to
effective treatment and also limit excessive spreading of drugs in other regions of the body.
Release rate study was carried out using high performance liquid chromatography (HPLC). Invitro and in-vivo study was carried out to check the efficacy of treatment.
Keywords: Targeted drug delivery, magnetite nanoparticle, breast cancer
vi
TABLE OF CONTENTS
Chapter
page
1.
INTRODUCTION ...............................................................................................................1
2.
LITERATURE REVIEW ....................................................................................................4
3.
FABRICATION AND EXPERIMENTAL METHODS .....................................................9
3.1
3.2
3.3
3.4
3.5
3.6
Materials .....................................................................................................................9
Fabrication of Magnetic Nanoparticle ........................................................................9
Synthesis of Microsphere ..........................................................................................11
Separation of Nanocomposite Sphere .......................................................................13
Attenuated Total Reflectance (ATR) Spectroscopy of Nanocomposite Sphere .......14
In-vitro Tests .............................................................................................................14
3.6.1 In-vitro Test using 3T3 Cells ...........................................................................14
3.6.2 In-vitro Test using Breast Cancer Cells (MDA 486) .......................................16
3.7 High Performance Liquid Chromatography .............................................................17
3.8 In-vivo Test ...............................................................................................................20
4.
RESULTS AND DISCUSSIONS ......................................................................................22
4.1
4.2
4.3
4.4
4.5
5.
Characterization of Microsphere...............................................................................22
In-vitro Test
Release Profile from High Performance Liquid Chromatography ...........................26
In-vivo Study ............................................................................................................32
Histology Study ........................................................................................................36
CONCLUSION AND FUTURE WORK ..........................................................................42
REFERENCES ..............................................................................................................................44
APPENDICES ...............................................................................................................................48
A.........................................................................................................................................49
B. ........................................................................................................................................76
C .........................................................................................................................................90
vii
LIST OF TABLES
Table
page
1.
Data for Calibration Chart of Cyclophosphamide in PBS .................................................67
2.
Data for Concentration of Cyclophosphamide in the PBS Sample ...................................67
3.
Cumulative Concentration of Cyclophosphamide in PBS Sample ....................................73
4.
Data for Calibration Chart of Cyclophosphamide in Acetonitrile .....................................74
5.
Data for Cyclophosphamide from 100mg/ml Suspension of Drug Carrier in
Acetonitrile ........................................................................................................................74
6.
Data for Cumulative Percentage Release of Cyclophosphammide in PBS .......................75
7.
Data for Tumor Size at Various Time Intervals.................................................................87
8.
Data for In-vitro Test with 3T3 Cells ................................................................................88
9.
Data for In-vitro Test with Breast Cancer Cells ................................................................89
10
Data for Percentage Live Cancer Cells in Tumors ............................................................98
viii
LIST OF FIGURES
Figure
page
1.
Some of the materials for PLGA microsphere fabrication ..................................................9
2.
Addition of ammonium hydroxide solution .......................................................................10
3.
Formation of magnetite nanoparticles ...............................................................................11
4.
Schematic for magnetite nanoparticle synthesis ................................................................11
5.
Oil in oil emulsion /solvent evaporation technique ...........................................................13
6.
Overhead mixer at 8500 rpm .............................................................................................13
7.
Plate for in-vitro test with 3T3 cells after MTT test ..........................................................16
8.
Plate for in-vitro test with breast cancer cell before incubation ........................................17
9.
Schematic for working principle of HPLC ........................................................................18
10.
HPLC system used to measure the release profile of cyclophosphamide from
microspheres ......................................................................................................................19
11.
Tumor on mouse and application of magnetic field ..........................................................21
12
Molecular configuration of (a) 5-FU and (b) Cyclophosphamide .....................................22
13
Molecular configuration of PLGA .....................................................................................23
14.
ATR spectrum of nanocomposite sphere ...........................................................................24
15.
In-vitro test result using 3T3 cells .....................................................................................25
16.
In-vitro test result using MDA 486 breast cancer cells......................................................26
17.
Calibration chart of peak area versus concentration of cyclophosphamide in PBS ..........27
18.
HPLC peak for ctclophosphamide in PBS having 10 mg/ml concentration ......................27
19.
Cumulative release profile of cyclophosphamide from microsphere ................................28
20.
HPLC peak for cyclophosphamide in the sample collected after 1 hour ...........................28
ix
LIST OF FIGURES (Continued)
Figure
page
21.
Calibration chart of peak area versus concentration of
cyclophosphamide in acetonitrile ......................................................................................29
22.
HPLC peak for cyclophosphamide in acetonitrile having 10mg/ml concentration ...........30
23.
Cumulative percentage release of cyclophosphamide from microsphere ..........................30
24.
HPLC peak for cyclophosphamide in collected supernatant
with acetonitrile as solvent.................................................................................................31
25.
Release mechanism from PLGA microsphere ...................................................................32
26.
Variation of tumor size at different time intervals .............................................................33
27.
Variation of tumor size at dufferent time intervals for the groups treated with
microspheres containing chemotherpeutics .......................................................................34
28.
Tumor on mouse from group 1(untreated) (a) Day 1
(b) Day 3 (c) Day 10 (d) Day 17 ........................................................................................34
29.
Tumor on mouse from group 2 (treated with pure chemotherpeutics)
(a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ........................................................................34
30.
Tumor on mouse from group 3 (treated with microspheres containing no drugs) (a) Day 1
(b) Day 3 (c) Day 10 (d) Day 17 ........................................................................................35
31.
Tumor on mouse from group 4 (treated with microspheres containing drugs) (a) Day 1 (b)
Day 3 (c) Day 10 (d) Day 17 .............................................................................................35
32.
Tumor on mouse from group 5 (treated with magnetic field and microspheres containing
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ............................................................35
33.
Necrotic site on a mouse from group 4 (treated with microspheres containing drugs) .....36
34.
Left side tumor sections of different mice from group 1 (untreated)
at 10X magnification..........................................................................................................37
35.
Tumor section of different mice from group 2 (treated with pure chemotherapeutic) at
10X magnification (a) Left side (b) Right side ..................................................................37
x
LIST OF FIGURES (Continued)
Figure
page
36.
Tumor sections of different mice from group 4 (treated with microspheres containing
drugs) at 10X magnification (a) Left side (b) Right side ...................................................38
37.
Tumor sections of different mice from group 5 (treated with microspheres containing
drugs + magnetic field) at 10X magnification (a) Right side (b) Right side .....................38
38.
Tumor sections of different mice from group 3 (treated with microspheres
containing no drugs) at 10X magnification (a) Left side (b) Right side ...........................39
39.
Left side tumor section of a mouse from group 1 (untreated) at 4X magnification ..........40
40
Right side tumor section of a mouse from group 4 (treated with chemotherpeutics) at 4X
magnification .....................................................................................................................40
41.
Variation of percentage live cells for different treatments ................................................41
42.
HPLC peak for solution of 10 mg/ml of cyclophosphamide in PBS for first run .............49
43.
HPLC peak for solution of 10 mg/ml of cyclophosphamide in PBS for second run .........49
44.
HPLC peak for solution of 5 mg/ml of cyclophosphamide in PBS for first run ...............50
45.
HPLC peak for solution of 5 mg/ml of cyclophosphamide in PBS for second run ...........50
46.
HPLC peak for solution of 2.5 mg/ml of cyclophosphamide in PBS for first run ............51
47.
HPLC peak for solution of 2.5 mg/ml of cyclophosphamide in PBS for second run ........51
48.
HPLC peak for solution of 1.25 mg/ml of cyclophosphamide in PBS for first run ..........52
49.
HPLC peak for solution of 1.25 mg/ml of cyclophosphamide in PBS for second run ......52
50.
HPLC peak for solution of 0.625 mg/ml of cyclophosphamide in PBS for first run ........53
51.
HPLC peak for solution of 0.625 mg/ml of cyclophosphamide in PBS for second run ....53
52.
HPLC peak for 1 hour sample for first run ........................................................................54
53.
HPLC peak for 1 hour sample for second run ...................................................................54
54.
HPLC peak for 2 hour sample for first run ........................................................................55
xi
LIST OF FIGURES (Continued)
Figure
page
55.
HPLC peak for 2 hour sample for second run ...................................................................55
56.
HPLC peak for 4 hour sample for first run ........................................................................56
57.
HPLC peak for 4 hour sample for second run ...................................................................56
58.
HPLC peak for 8 hour sample for first run ........................................................................57
59.
HPLC peak for 8 hour sample for second run ...................................................................57
60.
HPLC peak for 12 hour sample for first run ......................................................................58
61.
HPLC peak for 12 hour sample for second run .................................................................58
62.
HPLC peak for 24 hour sample for first run ......................................................................59
63.
HPLC peak for 24 hour sample for second run .................................................................59
64.
HPLC peak for 72 hour sample for first run ......................................................................60
65.
HPLC peak for 72 hour sample for second run .................................................................60
66.
HPLC peak for 10 mg/ml cyclophosphamide in acetonitrile for first run .........................61
67.
HPLC peak for 10 mg/ml cyclophosphamide in acetonitrile for second run ....................61
68.
HPLC peak for 5 mg/ml cyclophosphamide in acetonitrile for first run ...........................62
69.
HPLC peak for 5 mg/ml cyclophosphamide in acetonitrile for second run ......................62
70.
HPLC peak for 2.5 mg/ml cyclophosphamide in acetonitrile for first run ........................63
71.
HPLC peak for 2.5 mg/ml cyclophosphamide in acetonitrile for second run ...................63
72.
HPLC peak for 1.25 mg/ml cyclophosphamide in acetonitrile for first run ......................64
73.
HPLC peak for 1.25 mg/ml cyclophosphamide in acetonitrile for second run .................64
74.
HPLC peak for 0.625 mg/ml cyclophosphamide in acetonitrile for first run ....................65
xii
LIST OF FIGURES (Continued)
Figure
page
75.
HPLC peak for 0.625 mg/ml cyclophosphamide in acetonitrile for second run ...............65
76.
HPLC peak of cyclophosphamide for 100 mg/ml drug carrier in
acetonitrile for first run ......................................................................................................66
77.
HPLC peak of cyclophosphamide for 100 mg/ml drug carrier in acetonitrile for second
run ......................................................................................................................................66
78.
Left side tumor on mouse (723) from group 1(untreated) (a) Day 1
(b) Day 3 (c) Day 10 (d) Day 17 ........................................................................................76
79.
Right side tumor on mouse (723) from group 1(untreated) (a) Day 1 (b) Day 3 (c) Day 10
(d) Day 17 ..........................................................................................................................76
80.
Left side tumor on mouse (724) from group 1(untreated) (a) Day 1 (b) Day 3 (c) Day 10
(d) Day 17 ..........................................................................................................................76
81
Right side tumor on mouse (724) from group 1(untreated) (a) Day 1
(b) Day 3 (c) Day ...............................................................................................................77
82.
Left side tumor on mouse (725) from group 1(untreated) (a) Day 1 (b) Day 3 .................77
83.
Right side tumor on mouse (724) from group 1(untreated) (a) Day 1 (b) Day 3 ..............77
84.
Left side tumor on mouse (726) from group 1(untreated) (a) Day 1
(b) Day 3 (c) Day 10 (d) Day 17 ........................................................................................78
85.
Right side tumor on mouse (726) from group 1(untreated) (a) Day 1
(b) Day 3 (c) Day 10 (d) Day 17 ........................................................................................78
86
Left side tumor on mouse (727) from group 2 (treated with pure chemotherpeutics) (a)
Day 1 (b) Day 3 (c) Day 10 (d) Day 17 .............................................................................78
87.
Right side tumor on mouse (727) from group 2
(treated with pure chemotherpeutics) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ............78
88.
Left side tumor on mouse (728) from group 2 (treated with pure chemotherpeutics)
(a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ........................................................................79
89.
Right side tumor on mouse (728) from group 2 (treated with pure chemotherpeutics)
(a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ........................................................................79
xiii
LIST OF FIGURES (Continued)
Figure
page
90.
Left side tumor on mouse (729) from group 2 (treated with pure chemotherpeutics)
(a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ........................................................................79
91.
Right side tumor on mouse (729) from group 2 (treated with pure chemotherpeutics)
(a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ........................................................................79
92.
Left side tumor on mouse (730) from group 2 (treated with pure chemotherpeutics)
(a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ........................................................................80
93.
Right side tumor on mouse (730) from group 2 (treated with pure chemotherpeutics)
(a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ........................................................................80
94.
Left side tumor on mouse (731) from group 3 (treated with microsphere without drugs)
(a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ........................................................................80
95.
Right side tumor on mouse (731) from group 3 (treated with microsphere without drugs)
(a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ........................................................................80
96.
Left side tumor on mouse (732) from group 3 (treated with microsphere without drugs)
(a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ........................................................................81
97.
Right side tumor on mouse (732) from group 3 (treated with microsphere without drugs)
(a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ........................................................................81
98.
Left side tumor on mouse (733) from group 3 (treated with microsphere without drugs)
(a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ........................................................................81
99.
Right side tumor on mouse (733) from group 3 (treated with microsphere without drugs)
(a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ........................................................................81
100.
Left side tumor on mouse (734) from group 3 (treated with microsphere without drugs)
(a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ........................................................................82
101.
Right side tumor on mouse (734) from group 3 (treated with microsphere without drugs)
(a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ........................................................................82
102.
Left side tumor on mouse (735) from group 4 (treated with microsphere containing
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ............................................................82
103.
Right side tumor on mouse (735) from group 4 (treated with microsphere containing
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ............................................................82
xiv
LIST OF FIGURES (Continued)
Figure
page
104.
Left side tumor on mouse (736) from group 4 (treated with microsphere containing
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ............................................................83
105.
Right side tumor on mouse (736) from group 4 (treated with microsphere containing
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ............................................................83
106.
Left side tumor on mouse (737) from group 4 (treated with microsphere containing
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ............................................................83
107.
Right side tumor on mouse (737) from group 4 (treated with microsphere containing
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ............................................................83
108.
Left side tumor on mouse (738) from group 4 (treated with microsphere containing
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ............................................................84
109.
Right side tumor on mouse (738) from group 4 (treated with microsphere containing
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ............................................................84
110.
Left side tumor on mouse (739) from group 5 (treated with microsphere containing drugs
+ magnetic field) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ...........................................84
111.
Right side tumor on mouse (739) from group 5 (treated with microsphere containing
drugs + magnetic field) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 .................................84
112.
Left side tumor on mouse (740) from group 5 (treated with microsphere containing drugs
+ magnetic field) (a) Day 1 (b) Day 3................................................................................85
113.
Right side tumor on mouse (740) from group 5 (treated with microsphere containing
drugs + magnetic field) (a) Day 1 (b) Day 3 ......................................................................85
114.
Left side tumor on mouse (741) from group 5 (treated with microsphere containing drugs
+ magnetic field) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 ...........................................85
115.
Right side tumor on mouse (741) from group 5 (treated with microsphere containing
drugs + magnetic field) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17 .................................86
116.
Left side tumor on mouse (742) from group 5 (treated with microsphere containing drugs
+ magnetic field) (a) Day 1 (b) Day 3................................................................................86
117.
Right side tumor on mouse (742) from group 5 (treated with microsphere containing
drugs + magnetic field) (a) Day 1 (b) Day 3 ......................................................................86
xv
LIST OF FIGURES (Continued)
Figure
page
118.
Tumor sections at 4X magnification of the same mouse (723) from group 1
(untreated) (a) Left side (b) Right side ..............................................................................90
119.
Tumor sections at 4X magnification of the same mouse (724) from group
1(untreated) (a) Left side (b) Right side ............................................................................90
120.
Tumor sections at 4X magnification of the same mouse (726) from group 1
(untreated) (a) Left side (b) Right side ..............................................................................91
121.
Tumor sections at 4X magnification of the same mouse (727) from group 2
(treated with pure chemotherapeutics) (a) Left side (b) Right side ...................................91
122.
Tumor sections at 4X magnification of the same mouse (728) from group 2
(treated with pure chemotherapeutics) (a) Left side (b) Right side ...................................92
123.
Tumor sections at 4X magnification of the same mouse (729) from group 2
(treated with pure chemotherapeutics) (a) Left side (b) Right side ...................................92
124.
Tumor sections at 4X magnification of the same mouse (730) from group 2
(treated with pure chemotherapeutics) (a) Left side (b) Right side ...................................93
125.
Tumor sections at 4X magnification of the same mouse (731) from group 3
(treated with microspheres containing no drugs) (a) Left side (b) Right side ...................93
126. Tumor sections at 4X magnification of the same mouse (732) from group 3(treated with
microspheres containing no drugs) (a) Left side (b) Right side.....................................................94
127. Tumor sections at 4X magnification of the same mouse (733) from group 3(treated with
microspheres containing no drugs) (a) Left side (b) Right side.....................................................94
128. Tumor sections at 4X magnification of the same mouse (734) from group 3(treated with
microspheres containing no drugs) (a) Left side (b) Right side.....................................................95
129. Tumor sections at 4X magnification of the same mouse (735) from group 4(treated with
microspheres containing drugs) (a) Left side (b) Right side..........................................................95
130. Tumor sections at 4X magnification of the same mouse (736) from group 4(treated with
microspheres containing drugs) (a) Left side (b) Right side..........................................................96
131. Tumor sections at 4X magnification of the same mouse (737) from group 4(treated with
microspheres containing drugs) (a) Left side (b) Right side..........................................................96
xvi
LIST OF FIGURES (Continued)
Figure
page
132. Right side tumor section at 4X magnification of the same mouse (738) from group
4(treated with microspheres containing drugs) ..............................................................................97
133. Right side tumor section at 4X magnification of the same mouse (739) from group
5(treated with magnetic field and microspheres containing drugs) ...............................................97
134. Right side tumor section at 4X magnification of the same mouse (741) from group
5(treated with magnetic field and microspheres containing drugs) ...............................................98
xvii
LIST OF ABBREVIATIONS
PLGA
Poly(lactic-co-glycolic) Acid
5-Fu
5-Fluoroucil
HPLC
High Performance Liquid Chromatography
ATR
Attenuated Total Reflectance
OD
Optical Density
xviii
LIST OF SYMBOLS
µm2
Micrometer Square
˚C
Degree Celsius
M
Molarity
mg
Milligram
ml
Milliliter
mVolts
Millivolts
Sec
Seconds
xix
CHAPTER 1
INTRODUCTION
The clinical success of any treatment depends on the bioviability of drugs which includes
the route of drug administration, drug absorption rate and its metabolism in the body. Targeted
drug delivery is localized drug delivery to a specific disease site so that there will be interaction
of drugs only with disease tissue. Hence targeted drug delivery reduces side effects and improves
bioviability. Targeted drug delivery is very important in cancer treatment because of the toxicity
of chemotherapeutics.
Nanotechnology has opened various paths of treatment methods for cancer therapy. It
involves a wide variety of fields such as engineering, materials science, chemistry and physics
along with cancer biology. Extensive research is going on to improve the efficacy of treatment
with targeted drug delivery. The size of carriers used for cancer drug delivery is such that they
are able to pass through the leaky endothelium of cancer cells. Drug carriers accumulate in the
tumor site by means of active or passive process. The passive transport mainly involves
enhanced permeability and retention (EPR) effect. For tumor cells to grow rapidly there is
enhanced growth of blood vessels around tumor cells. In order to meet the growing need of
nutrition and oxygen of cancer cells, new blood vessels form. These blood vessels are abnormal
in architecture with wide lumen. Tumors also possess poor lymphatic system. All these lead to
excessive molecule and fluid transport. As a result EPR effect plays an important role in carrying
drug molecules or nanoparticles to tumor cells. Active transport can be achieved to the desired
site by means of molecular recognition. Drug carriers can be functionalized to drive the particles
to the targeted site. There are many ways of achieving targeted drug delivery. There can be pH
1
sensitive drug carriers which release drugs at lower pH which is characteristics of tumor.
Ultrasound can cause drug loaded vehicles to release drugs at a specific site. Thermally
responsive carriers accumulate and release drugs in heated solid tumors. The drug loaded carrier
can be magnetized and it can be driven and retained at the desired site using external magnetic
field of appropriate strength. Photosensitive drug carriers are designed to release drugs when
they are exposed to electromagnetic wave of certain wavelength. Drug carriers can be
functionalized with ligands or antibodies which will direct them to specific tumor receptors.
There are many types of nanocarriers such as dendrimers, micelles and liposomes.
Dendrimers possess three dimensional tree-like structures with multifunctional core molecule at
the center. Drug molecules can be attached to its functional groups and its branches can be
functionalized with ligands or antibodies specific to a tumor. Micelles are spherical in nature.
They possess both hydrophobic and hydrophilic end. The hydrophilic end is towards the outer
surface and the hydrophobic tails face towards the inside of micelles. Liposomes are vesicles
with lipid layers. There are two kinds of it, unilamellar and multilamellar. Unilamellar liposomes
possess aqueous core and hence are able to carry water soluble drugs. Multilamellar liposomes
can carry lipid soluble drugs. Liposomes have a polymeric coating which prevent its
disintegration and hence give it steric stability. Drug molecules can be entrapped or attached to
nanospheres for drug delivery purpose. Nanocapsules have central cavity for drug entrapment
and its core can have aqueous or oily atmosphere.
In this research work, chemotherapeutics, magnetic nanoparticles, fluorescence tag and
albumin loaded microsphere was used for the treatment of breast cancer. The microsphere is
made of biodegradable poly(D, L-lactide-co-glycolide) (PLGA). Albumin and magnetic
nanoparticles are used to obtain targeted delivery. External magnetic field is used to guide and
2
accumulate microspheres in the tumor. Albumin itself drives the carriers towards tumor cells as
tumor cells draw albumin for nutrition purpose. Use of polymeric microsphere has allowed
controlled release of drugs. A combination of two types of drugs are used which are 5Fluorouracil (5-Fu) and cyclophosphamide. Characterization of the drug delivery system was
done using attenuated total reflectance (ATR) spectroscopy. The release profile of drug was
studied using high performane liquid chromatography. In-vitro and in-vivo study was carried out
to observe the efficacy of treatment against breast cancer.
3
CHAPTER 2
LITERATURE REVIEW
A lot of research is dedicated towards targeted drug delivery especially for cancer
treatment. Most of these researches involve development of novel multifunctional drug delivery
vehicles. Magnetic nanoparticles have opened novel pathways towards targeted drug delivery
system, hyperthermia, magnetic resonance imaging and many other clinical applications [1-3].
Magnetic targeted drug delivery holds promising future for cancer treatment. Extensive research
involving development of high momentum magnetic nanoparticle carriers is underway to
overcome the constraints of magnetic targeted drug delivery such as decrease of magnetic field
strength with increasing depth of target site [4].
Takeda et al successfully guided ferromagnetic particles through Y-shaped glass tubes by
means of superconducting magnet [5]. The magnet was placed at a distance from the tube that is
comparable with the distance between rat‟s vena cava and body surface. Ciofani et al developed
alginate magnetic nanoparticles and loaded them into bovine serum albumin [6]. He used a
bioreactor having two cell culture compartments to conduct experiments. He used a permanent
magnet to accumulate nanoparticles in one compartment successfully. Mu et al developed
magnetic hybrid emulsion droplets [7]. He fabricated polyanion/polycation multilayerencapsulated oil in water hybrid emulsion droplet cores. Drug molecules were loaded in the
hybrid emulsion droplet. A controlled release profile was observed at pH 1.8. Goodrazi et al.
fabricated homogeneous aqueous iron oxide magnetic nanoparticles and functionalized its
surface with citric acid [8]. The carboxylate group provides possibility for further
functionalization to be used as targeting and bioimaging agents. PLGA microparticles containing
4
magnetite nanocrystals and insulin were directed to gastrointestine by means of external
magnetic field [9]. It was seen that there was substantial reduction in blood glucose level when
these microparticles were administered in mice. The average microparticle size for 0, 2 and 5
wt% of magnetite was 4.6, 6.4 and 7.2 µm respectively. Insulin encapsulation efficiency was 68,
78 and 79% for the mentioned contents of magnetite. Water-in-oil-in water solvent evaporation
technique was employed to produce the PLGA microparticles. Iron oxide magnetic nanoparticle
was functionalized with Gum arabic [10]. Rhodamine B was grafted on the magnetic
nanoparticle (RDB-GAMNP) and dexamethasone drug was conjugated to the RDB-GAMNP by
means of photosensitive linker. Drug release was triggered by light source. Rutnakornpituk et al
synthesized a drug loaded magnetic nanoparticle of approximately 9 nm in diameter [11]. The
particle was coated with oleic acid followed by poly (ethylene glycol) methyl ether-poly (εcaprolactone) (mPEG-PCL). The particle had PCL hydrophobic inner shell and mPEG
hydrophilic corona. It showed superparamagnetic behavior at room temperature with tunable
drug release profile. Drug loaded magnetic nanocomposite spheres were fabricated using
magnetite
nanoparticles
and
poly(D,L-lactide-co-glycolide)
(PLGA)
by
oil-in-oil
emulsion/solvent evaporation technique [12]. The nanocomposite spheres containing magnetite
were approximately 200 nm to 1.3 µm in diameter and they exhibited superparamagnetic
behavior above the blocking temperature. This shows these nanocomposite spheres are potential
drug delivery vehicle for magnetic targeted drug delivery. Asmatulu et al. reported influence of
various parameters on magnetic guidance [13] Oh et al synthesized carbonized polypyrrole by
pyrolysis of polypyrrole nanoparticles which was made by micelle templating in oil/water
emulsion [14]. The nanoparticles were made to undergo heat treatment during when the
nanoparticles turned into carbon nanoparticles and the doped iron cations into magnetic phase.
5
He studied the release profile of ibuprofen from these particles and observed that the particles
exhibited sustained slow release because of higher surface area and pore volume. The particles
possessed low toxicity when its concentration was lower than 100 µm/ml. Miscelle size is
dependent on concentration of surfactant. Miscelle size decreases with increasing concentration
of surfactant. The polypyrrole nanoparticle size was controlled by varying the concentration of
DTAB and decyl alcohol. Avilé et al used magnetic implant to capture magnetic drug carrier
particles [15]. Dexatran coated magnetic particles were used for seeding and polydivinylbenzene
magnetite particles acted as magnetic drug carrier particles. A porous polyethylene cylinder was
used as tissue capillary. A permanent magnet at a specific distance from the cylinder was used to
capture the seeding particles which in turn increased the capturing efficiency of magnetic drug
carrier particles. Thus it is possible to improve drug delivery from magnetic drug carrying
vehicles at the targeted site which is deep within the body using magnetic implants. Rodrigo et al
studied the use of permanent magnet as implant in the target organ to improve the efficacy of
magnetic targeted drug delivery of anti-cancer drugs [16]. Wilson et al performed clinical study
on four patients using magnetic targeted drug delivery and MRI [17]. In his study hepatocellular
carcinoma was treated with magnetically targeted drug carrier having doxorubicin (MTC-DOX).
A 5 kg magnet was placed on the patient‟s abdomen closest to the tumor to draw the drug
carriers from vascular space to surrounding tumor tissues. Gelatin magnetic microspheres were
synthesized by emulsification/cross-linking method [18]. Diclofenac sodium was loaded into it
and it was delivered by intra-arterial injection to the target site using external magnet.
Release profile of drugs from drug carriers is very important. Porous poly(lactic-coglycolic acid) (PLGA) microspheres were fabricated to load simvastatin (SIM) drug [19].
Approximately 80% SIM encapsulation efficiency was achieved. About 18% of drug was
6
released between 1 to 2 days and then slow sustained release was observed. Lin et al
encapsulated doxorubicin in PLGA microparticles by spray drying technique [20]. He improved
the release characteristics of drug by using pluronic (PLU) and poly (L-Lactide) (PLLA) along
with PLGA. Degradation rate of PLGA was reduced when the lactide/glycolide ratio was
increased. PLLA/PLGA composite microparticles exhibited reduced release profile as PLLA
formed a protective layer preventing fast release of drugs. Wei et al fabricated PLGA
microspheres and loaded them with bovine serum albumin or human parathyroid hormone (PTH)
(1-34) [21]. The microspheres exhibited high encapsulation efficiency. He studied the
degradation, protein loading and release kinetics of the PLGA 50:50 microspheres. The
microspheres degraded rapidly after 3 weeks and there was detectable amount of PTH release
within 24 hours.
Studies have shown enhanced uptake and retention of albumin in tumor tissues [22, 23].
Stehle et al suggested that tumor nutrition is based on excessive plasma protein catabolism and
that albumin can be a key source of nutrition for tumor proliferation [23]. Albumin is stable in
pH 4-9, soluble in 40% ethanol and able to sustain itself at 60˚C for 10 hours [22]. It is
preferentially taken up by tumor tissues and it is also biodegradable and non-toxic [22]. All these
features make albumin a perfect drug delivery carrier for tumor treatment. Because of the same
reasons albumin was used as a carrier to deliver methotrexate (MTX) for the treatment of
rheumatoid arthritis [24]. Efficiency of treatment with MTX coupled to human serum albumin
(HSA) in arthritic mice was studied [24]. It was found that MTX coupled with HSA exhibited
effective treatment against arthritis compared to MTX only. Agarwal et al studied the use of
MTX loaded solid-lipid nanoparticle conjugated with HSA to deliver MTX to brain [25]. He also
observed accumulation of the conjugate in high concentration in cancer cells compared to plain
7
solid-lipid nanoparticle. Tumors and inflammatory cells harness amino acids and energy by
metabolizing albumin [26]. Human serum albumin contains various functional groups on its
hydrophilic surface as well as in hydrophobic binding pockets [26]. Thus hydrophobic molecules
and drugs can bind to the hydrophobic binding sites of albumin molecule. Therefore albumin is a
potential carrier to deliver drugs at target sites like tumor and inflammatory disease sites.
It is also possible to achieve targeted drug delivery by means of targeting ligands. Alexis
et al used anti-[human epidermal growth factor receptor 2] (HER-2) affibody as targeting ligand
[27]. He conjugated anti-HER-2 affibody to poly (D, L lactic acid)-poly (ethylene glycol)maleimide (PLA-PEG-Mal) copolymer nanoparticle and encapsulated paclitaxel in it. This
carrier can be used for drug delivery to HER-2 positive cell lines like SKBR-3 and SKOV-3.
Aptamers are outstanding substitute to antibodies for targeted drug delivery. Cisplatin
encapsulated liposome having aptamer conjugation have been reported [28]. Aptamer derived
from AS1411 was used which possessed high affinity to nucleolin (NCL). The latter is
overexpressed in various human disease sites such as breast cancer. Gold nanoparticles
conjugated with aptamers can be used to detect cancer cells [29]. The aptamer used specifically
bind to platelet derived growth factor (PDGF) which were overexpressed in certain cancer cells.
This shows the potential of aptamers to be used as targeting ligands.
In this research work a multifunctional novel microsphere made from poly(D L-lactideco-glycolide) (PLGA) has been developed for targeted drug delivery. It is loaded with two types
of chemotherapeutics, cyclophosphamide and 5-Fluorouracil (5-Fu). It also contains magnetite
nanoparticles and albumin for targeting purpose. Fluorescent agent is added to it for imaging.
The microsphere has been characterized for all the components and its release rate is studied. Invitro and in-vivo tests are done to evaluate the efficacy of treatment against breast cancer.
8
CHAPTER 3
FABRICATION AND EXPERIMENTAL METHODS
3.1 Materials
Poly(D L-lactide-co-glycolide) (PLGA 50:50) (MW 7000-17000), Albumin from human
serum (96-99%), Cyclophosphamide Monohydrate, 5-Fluorouracil minimum 99% TLC, 1,6Diphenyl-1,3,5-hexatriene 98%, Petroleum Ether Spectrophotometric Grade, Paraffin Oil,
Ammonium Hydroxide were purchased from Sigma Aldrich. Figure 1 shows some of the
materials used in the fabrication process. Hexanes Optima, Hydrochloric Acid (normality 12.1)
were purchased from Fisher Scientific and Acetonitrile from ACROS ORGANICS. Span-80 was
purchased from Fluka. Iron (II) chloride and iron (III) chloride were bought from Alfa Aesar.
Figure 1. Some of the materials for PLGA microsphere fabrication
3.2 Fabrication of Magnetic Nanoparticle
A solution of 1g iron (II) chloride (FeCl2.4H2O) in 25ml of 2M hydrochloric acid (HCl) was
prepared.
Another solution of 1.6g iron (III) chloride (FeCl3.6H2O) in 100ml of 2M
hydrochloric acid (HCl) was made. Then these two solutions were mixed in a beaker and stirred
with a magnetic bar for 10 minutes. After this 25ml of this solution was transferred to another
beaker and stirred vigorously. During stirring 6ml of 30% ammonium hydroxide solution was
9
added dropwise by a syringe in 5 minutes. Figure 2 shows the drop wise addition of ammonium
hydroxide solution. There would be formation of black nanoparticles as shown in figure 3. The
magnetic stirring bar was removed and a strong Nd magnet was held underneath the beaker so
that the black particles would settle down. The top liquid was decanted. Then 25 ml of DI water
was added to prevent nucleation and growth. The Nd magnet was held underneath the beaker and
when the particles settled down, water was decanted. This process was repeated five times to
wash the particles. The resulting particles were iron oxide magnetite nanoparticles of
approximately 10 nm in size. The schematic diagram for synthesis of magnetite nanoparticles are
shown in figure 4.
2FeCl3 + FeCl2 + 8NH3 + 4H2O = Fe3O4 + 8NH4Cl
Figure 2. Addition of ammonium hydroxide solution
10
Figure 3. Formation of magnetite nanoparticles
FeCl2
solution in
2M HCl
FeCl3
solution in
2M HCl
30%
ammonium
hydroxide
solution
Magnetite Nanoparticle
Figure 4. Schematic for magnetite nanoparticle synthesis
3.3 Synthesis of Microsphere
Magnetic nanocomposite spheres were fabricated using Poly(D L-lactide-co-glycolide)
(PLGA) and magnetic nanoparticles by oil-in-oil emulsion/solvent evaporation technique. The
synthesis process consisted of two phases. First phase consisted of an acetonitrile system. In this
phase required percentage of magnetite nanoparticle (13.5%) was added to 5ml acetonitrile and
sonicated for 10 minutes with a probe sonicator in order to disperse it properly. Then 1.25% w/v
11
PLGA was added to the 5 ml solvent and sonicated for 5 minutes. Once PLGA dissolved, anticancer drugs (cyclophosphamide and 5-FU), albumin and fluorescent tag (1,6-Diphenyl-1,3,5hexatriene) were added and the whole mixture was sonicated for another 15 minutes.
The second phase consisted of paraffin oil and span-80 emulsifier. This phase was made
by adding 0.2ml span-80 as a surfactant to 40ml of paraffin oil. The mixture was agitated by an
overhead mixer at 8500 rpm. Then phase 1 was added dropwise to phase 2 and the mixture of the
two phases were agitated for one and a half hour so that acetonitrile would evaporate and
nanocomposite sphere would form.
Figure 5 and 6 shows the schematic diagram for the fabrication process of nanocomposite
sphere and the overhead mixer respectively. The composition of various components used is as
follows:
PLGA: 32%
Magnetite: 13.5%
Albumin: 28%
Cyclophosphamide: 12.5%
5-Fluorouracil (5-FU): 12.5%
Flourescence Tag: 1.5%
12
Figure 5. Oil in oil emulsion /solvent evaporation technique
Figure 6. Overhead mixer at 8500 rpm
3.4 Separation of Nanocomposite Sphere
The oil mixture was centifuged at 17000 rpm for 10 minutes at 10⁰C and the
nanocomposite spheres were collected. Then they were washed twice with hexane containing 5%
13
petroleum ether and twice again with hexane only. After this hexane containing 5% carbon
tetrachloride was added to the particles and sonicated in order to disperse the microspheres, then
the mixture was filtered with 0.2 micron paper cloth.
3.5 Attenuated Total Reflectance (ATR) Spectroscopy of Nanocomposite Sphere
In ATR system, a beam of infrared light is passed through its crystal so that there will be
reflection at the internal surface in contact with the sample. The reflection results in an
evanescent wave that travels through the sample. It is used to study chemistry of molecules. Its
working principal is based on the fact that molecules absorb certain frequencies that correspond
to their molecular structure. The frequency of the vibrating group or bond corresponds to the
absorbed frequency.
3.6 In-vitro Tests
This test gave insight about release profile of the drug carrier and its efficacy against
cancer treatment. In-vitro test is carried out using 3T3 cells and breast cancer cells (MDA-486).
The compositions of
various components of the drug carrrier are same as mentioned in
fabrication section.
3.6.1 In-vitro Test Using 3T3 Cells
20 mg of drug carrier was added to 1 ml of cell culture medium to make a suspension of
20 mg/ml and the suspension was incubated at 37°C. After 1 day incubation, the suspension was
centrifuged and supernatant was collected and refrigerated at -20°C. Then 1 ml of fresh cell
medium was added and it was incubated again. This process was repeated in order to collect
supernatant for day 3 and 5. Approximately 40,000 3T3 cells per well per 100 μl of cell medium
were cultured in 96 well plate. The plate was then incubated at 37°C overnight for cell
14
attachment to the well base. 500 μl of supernatant for day 1, 3 and 5 was added to separate tube
wells in the first column followed by mixing. Then 250 μl of fresh medium was added to each of
tube wells starting from second column. After this 250 μl of supernatant was trasferred from tube
wells in first column to the next adjacent tube wells and this was continued in order to achieve
1:2 dilution. 100 μl of supernatant of various concentrations for day 1, 3 and 5 were added to the
plate wells. The starting concentration for day 1, 3 and 5 was 2000 μg in 100 μl. After adding to
the wells containing 100 μl of cell medium, the concentration changed to 2000 μg in 200 μl or 10
mg/ml. So for wells in a row the concentrations were 10 mg/ml, 5 mg/ml, 2.5 mg/ml, 1.25
mg/ml, 0.625 mg/ml, 0.3125 mg/ml, 0.15625mg/ml, 0.078125 mg/ml, 0.0390625 mg/ml,
0.01953125 mg/ml, 0.009765625 mg/ml, and 0.004882813 mg/ml. The plate was incubated at
37°C for 7 days. For control experiment, two rows of the plate contained only fresh medium and
no supernatant. Then 20 μl of MTT (3-(4,5 –Dimethylthiazol-2-YI)-2,5-Diphenyltetrazolium
Bromide) was added to each well and the plate was incubated again at 37°C for 6 hours. After
incubation of 6 hours, all liquid from each well was carefully taken out using pipette. Then 200
μl of 10% sodium dodecyl sulfate (SDS) was added and the plate was incubated overnight. After
that 150 μl of SDS from each well is trasferred to a well of a new plate. The optical density (OD)
of the new plate is read at 590 nm. Figure 7 shows the plates for in-vitro test using 3T3 cells after
the MTT test.
15
Figure 7. Plate for in-vitro test with 3T3 cells after MTT test
3.6.2 In-vitro Test Using Breast Cancer Cells (MDA-486)
A suspension of 20mg/ml of drug carrier in cell medium was prepared as in the case of
in-vitro test with 3T3 cells. The suspension was incubated at 37°C and after day 1 it was
centrifuged. The supernatant was collected and stored in the refrigerater at -20°C and 1ml of
fresh medium replaced the supernatant. This was repeated in order to collect supernatant for day
3, 5 and 7. Breast cancer cell was cultured in 96 well plate such that there would be
approximately 20,000 cells per well in 100 μl of medium. The plate was incubated overnight for
cell attachment. Then 500 μl of supernatant for day 1, 3, 5 and 7 was added to separate tube
wells in the first column followed by mixing. After this 250 μl of fresh medium was added to
tube wells starting from second column. Liquid from tube wells in first column was transferred
to tubes in the next adjacent column. This was repeated to achieve 1:2 dilution. 100 μl of
supernatant of various concentrations for day 1, 3, 5 and 7 was added.to plate wells As discussed
above the concentration of liquid for wells in a row were 10 mg/ml, 5 mg/ml, 2.5 mg/ml, 1.25
mg/ml, 0.625 mg/ml, 0.3125 mg/ml, 0.15625mg/ml, 0.078125 mg/ml, 0.0390625 mg/ml,
16
0.01953125 mg/ml and 0.009765625 mg/ml. For control experiment, wells containing fresh
medium were used. The plate was incubated for 6 days. Then 20 μl of MTT was added to all
wells and the plate was incubated for 6 hours. The liquid from each well was removed by a
pipette and 200 μl of 10% SDS was added. The plate was incubated overnight. 150 μl of SDS
from each well is transferred to a well in new plate. The optical density was then read at 590 nm.
Figure 8 shows the plate for this in-vitro test before incubation.
Figure 8. Plate for in-vitro test with breast cancer cell before incubation
3.7 High Performance Liquid Chromatography
High performance liquid chromatography is a type of liquid chromatography which
separates components present in a solution. A solvent is forced through a column under high
pressure. For normal phase HPLC, the column consists of silica particles and the solvent used is
non-polar. When polar compounds pass through the column they become attracted to polar silica.
So their flow is hindered. In case of non-polar compounds they will flow through the column
quickly without hindrance. For reversed phase HPLC, the silica is made non-polar by means of
long hydrocarbon chains attached to silica surface. In this case a polar solvent is used. When
polar molecules pass through the column, there will be interaction with polar solvent but no
17
attraction to the non-polar silica which is the stationary phase. Therfore polar compounds will
flow through the column quickly. For non-polar molecules there will be attraction to the
hydrocarbon chains due to van-der-waals force. As a result they will travel slowly through the
column. Figure 9 shows a schematic of HPLC system.
Figure 9. Schematic for working principle of HPLC
The time taken by a specific compound to flow through column is known as retention time. The
retention time varies with various compounds. Apart from the type of compound, retention time
depends on pump pressure, features of stationary phase, solvent and column temperature. There
are many ways of detection but ultraviolet absorption is mostly used. The output is displayed as
peaks and each peak represents a specific compound passing through the column. The retention
time under controlled condition is unique for a specific compound and it can be used to identify a
compound. The area under the peak is proportional to the amount of the compound in the
injected sample.
18
Reversed phase HPLC was employed in this experiment to determine the release rate of
drug from carrier. „VARIAN ProStar‟ HPLC system was used. A suspension of 10 mg drug
carrier in 100 μl of 0.015M phosphate buffer (PBS) having pH 7.4 was prepared and left at room
temperature. Then at specific interval of time the suspension was centrifuged and 30 μl of
supernatant was collected and same amount of fresh PBS was replaced back into the suspension.
HPLC was carried out using the supernatant collected after various interval of time.
Cyclophosphamide was detected and the release rate of cyclophosphamide was determined.
Based on the methodology followed by Kensler et al, HPLC for detecting cyclophosphamide was
carried out [29]. The flow rate was set to 1 ml/min and sample volume injected was 10 μl. The
mobile phase consisted of 70% water and 30% acetonitrile. Ultraviolet absorbance was used as
the method of detection. The wavelength used was 205nm. Figure 10 shows the HPLC system
used for this experiment. The configuration of column used is given below:
Column: Bondclone 10µ C18,
10 micron
19
Figure 10. HPLC system used to measure the release profile of cyclophosphamide from
microspheres
3.8 In-vivo Test
This test was done in animal model to see the efficacy of the drug delivery system.
Twenty nude mice were divided into five groups. Each group went through following treatment:
Group 1: No treatment
Group 2: Treatment with pure drug (combination of 5-Fu and cyclophosphamide)
Group 3: Treatment with microspheres containing no drug
Group 4: Treatment with microspheres containing drug
Group 5: Treatment with microsheres containing drug and application of magnetic field
One million breast cancer cells in 0.5 ml of cell medium was injected into each side of a mouse
and allowed to grow for about a week. Figure 11(a) shows an image of tumor on a mouse before
treatment. For group 2, a solution of 1.25 mg/ml for each drug was prepared by dissolving 18.75
20
mg of 5-Fu and 18.75 mg of cyclophosphamide into 15 ml of PBS. For group 3,4 and 5, 2mg of
microspheres in 0.2 ml PBS was injected into each side of mouse near four points around tumor
for first injection. For second, third and fourth injection, 4mg microspheres in 0.2 ml of PBS was
injected into each side near four points around the tumor. For group 5 right after injection, small
magnet was attached to the tumor by means of glue as shown in figure 11(b). Tumor size was
observed for 17 days and then mice were sacrificed. After every two days the tumor size was
measured. Most of the tumors were irregular in shape. The length which is the longest
dimension, the width which is perpendicular to length and in the same plane and the height
which is the distance between mouse‟s body and outer edge of tumor were measured. The tumor
size was calculated using the formula below [31]:
(3.1)
According to Tomayko et al. the above formula correlates well with the mass of the tumor [31].
That is why it has been used to determine the tumor size.
Figure 11. (a) Tumor on a mouse before starting treatment (b) Application of magnetic field
using permanent magnet on tumor
21
CHAPTER 4
RESULTS AND DISCUSSIONS
4.1 Characterization of Microsphere
Attenuated total reflectance (ATR) spectroscopy of the nanocomposite sphere had been
employed to examine whether the expected components were present. Figure 12(a) and (b)
shows the molecular structure of 5-FU and cyclophosphamide respectively. The structure of
PLGA is exhibited by figure 13. It can be seen that 5-FU contains C-F, C-N, C-H, C=O, C=C, CC and N-H bonds. Cyclophosphamide possesses C-C, C-H, C-O, P=O, P-O, P-N, N-H, C-N and
C-Cl bonds. The structure of PLGA shows the presence of C-C, C=O, C-O and C-H bonds.
Figure 12. Molecular configuration of (a) 5-FU and (b) Cyclophosphamide
22
Figure 13. Molecular configuration of PLGA
The ATR spectroscopy of the nanocomposite sphere is shown in figure 14. At 460 cm-1 the
bending vibration of Fe-O bond occurs [32]. Also at 570 cm-1, the Fe-O bond vibration occurs.
When these are compared with the spectroscopy, peaks close to the above mentioned frequencies
can be seen showing the presence of iron oxide magnetic nanoparticles in the nanocomposite
sphere. The absorption frequencies for C-F and C-N are 960 to 1350 cm-1 and 1000 to 1350 cm1
respectively [33]. The spectroscopy shows peaks within these ranges as marked in figure 14.
This indicated that both these bonds are present in the nanocomposite sphere. The absorption
fraquency for C=C is between 1680 to 1640 cm-1 [33]. A clear peak at 1660.12 cm-1 can be seen
in the obtained IR spectrum and hence this bond also exists in the nanocomposite sphere. Since
C-F bond is only present in 5-FU, it can be said that 5-FU is present in the nanocomposite
sphere. The absoprtion frequency for P=O bond is 1280cm-1 and that for P-O bond is 1155 cm-1
[34]. The spectrum below shows peaks approximately at those frequencies and hence both these
bonds exist in the nanocomposite sphere indicating the presence of cyclophosphamide. The latter
also contains C-Cl bond which has absorption frequeny between 500-800 cm-1 [33]. The peak at
815.32 cm-1 shows its presence. PLGA and 5-FU both contain C=O which has absorption peak
23
between 1760 to 1670 cm-1 [33]. A clear peak can be observed at 1754.71 cm-1 showing the
existence of C=O bond.
C-F or
C-N
C-Cl
P=O
C=O
C=C
Fe-O
P---O
__
__
__
_
Figure 14. ATR spectrum of nanocomposite sphere
4.2 In-vitro Test
In-vitro test gave an estimation of drug release profile and also the concentration required
for effective treatment. Figure 15 shows in-vitro result for 3T3 cells. It can be seen that most of
the drug is released within day 3 as the OD reading is very low showing most of the cells are
dead. Compared to the control experiment where the wells contained only medium, OD reading
for day 5 is still lower showing some realease of drug.
24
In-Vitro Study on 3T3 cells
1.4
Optical Density
1.2
1
0.8
0.6
Day 1
0.4
Day 3
0.2
Day 5
0
Medium
Concentration (mg/ml)
Figure 15. In-vitro test result using 3T3 cells
It is also observed that OD readings for low concentrations of drugs are close to those of the
medium. This shows that the treatment is not effective at low concentrations. The OD readings
for day 3 and 5 are high and close to the corresponding readings for medium from concentrations
0.3125 mg/ml and below. The results using breast cancer cells are similar as shown in figure 16.
25
Optical Density
In-vitro study using MDA 486 breast cancer cells
4
3.5
3
2.5
2
1.5
1
0.5
0
Day 1
Day 3
Day 5
Day 7
Medium
Concentration (mg/ml)
Figure 16. In-vitro test result using MDA 486 breast cancer cells
4.3 Release Profile From High Performance Liquid Chromatography
The rate of cyclophosphamide release from drug carrier is determined using HPLC. In
order to determine the concentration of cyclophosphamide in the sample a calibration chart of
concentration versus peak area is made by running HPLC using authentic samples at known
concentrations. Figure 17 and 18 shows calibration graph and HPLC peak for cyclophosphamide.
26
Calibration chart for cyclophosphamide in PBS
3500
y = 300.34x
R² = 0.9742
Area (mVolts*sec)
3000
2500
2000
1500
1000
500
0
0
2
4
6
8
10
12
Concentration (mg/ml)
Figure 17. Calibration chart of peak area versus concentration of cyclophosphamide in PBS
Figure 18. HPLC peak for ctclophosphamide in PBS having 10 mg/ml concentration
The peak area for the sample and calibration chart are used to determine the concentration of
cyclophosphamide in the collected sample. Appendix A shows the HPLC reults for authentic as
well as experimental samples. Apendix A also shows the calculation for concentration of
27
cyclophosphamide in the collected sample at various time interval. Figure 19 and 20 exhibit the
cumulative release rate of cyclophosphamide and trypical HPLC peak respectively.
Cumulative Concentration (mg/ml)
Release of Cyclophosphamide
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
10
20
30
40
50
60
70
80
Time (hr)
Figure 19. Cumulative release profile of cyclophosphamide from microsphere
Figure 20. HPLC peak for cyclophosphamide in the sample collected after 1 hour
28
It can be seen from the release rate graph that drug is released very fast within 12 hours showing
burst release. Then the release rate slows down a lot. In order to calculate the percentage release
of drug at various time interval a solution of 10 mg drug carrier in 100 μl of acetonitrile was
prepared and the solution was vortexed. PLGA dissolved in acetonitrile releasing approximately
all the encapsulated drug. The solution was centrifuged and the supernatant was collected. HPLC
was carried out using the supernatant to determine the approximate total amount of drug in 10
mg of drug carrier. In order to determine the appriximate total amount of drug, a calibration chart
for various concentrations of cyclophosphamide in acetonitrile was prepared. Figure 21, 22, 23
and 24 exhibit calibration chart for cyclophosphamide in acetonitrile, typical HPLC peak for
authentic sample using acetonitrile as solvent, cumulative percentage release rate of
cyclophosphamide from drug carrier and typical HPLC peak for experimental sample in
acetonitrile.
Area (mVolts*sec)
Calibration chart for cyclophosphamide in
acetonitrile
4500
4000
3500
3000
2500
2000
1500
1000
500
0
y = 383.28x
R² = 0.9553
0
2
4
6
8
10
12
Concentration (mg/ml)
Figure 21. Calibration chart of peak area versus concentration of cyclophosphamide in
acetonitrile
29
Figure 22. HPLC peak for cyclophosphamide in acetonitrile having 10mg/ml concentration
Cumulative Percentage Release
Percentage Release of Cyclophosphamide
90
80
70
60
50
40
30
20
10
0
0
10
20
30
40
50
60
70
80
Time (hr)
Figure 23. Cumulative percentage release of cyclophosphamide from microsphere
30
Figure 24. HPLC peak for cyclophosphamide in collected supernatant with acetonitrile as solvent
It can be seen from the percentage release graph that approximately 77% cyclophosphamide is
released within 12 hours and rest of the drug is released very slowly. Figure 25 shows a
schematic of the release mechanism from PLGA microspheres. At first a burst release of drug
occurs due to drug on surface layer [20]. This is evident from the release profile. Then there will
be slow release mainly due to diffusion as can be seen from the release rate graph and finally
there will be second burst release due to rapid degradation of the microsphere [20]. The final
stage is not observed in the release profile graph because the release study was only for 72 hours.
Further study over long time period is required in order to see the last stage.
31
Figure 25. Release mechanism from PLGA microsphere
4.4 In-vivo Study
The in-vivo test was carried out using nude mice for 17 days and during this period the
tumor size was monitored. Figure 26 shows how the tumor size of various experimental groups
varied with time. For experimental group treated with microspheres containing no drug, the
tumor size increased continuously, showing that except for the chemotherapeutic all other
materials were not as toxic.as the drugs. For the group treated with pure chemotherapeutic, the
tumor size decreased only during the time of treatment that is during the first eight days. After
stopping the treatment tumor size increased again for this group. For groups treated with
microspheres containing drugs, tumor size followed a decreasing trend throughout the
experiment. For these two groups there were continuous release of drug from the microspheres
into the tumor. Drug is continuously released from the microspheres over a long period by
diffusion after first burst release. As a result cancer cells are exposed to chemotherapeutic over
longer period than the group treated with pure drug. The tumor size for the control group which
was not treated continued to increase.
32
Figure 26. Variation of tumor size at different time intervals
The tumor size for the group treated with microspheres containing drug and application of
magnetic field is slightly smaller when compared with tumor size of the group treated with
microspheres containing drugs and no application of magnetic field. This is shown in figure 27.
The reason for not observing a major difference was that particles were injected into the tumor
underneath the skin and they did not diffuse into the blood stream. Magnetic field will improve
the efficacy of treatment a lot when the particles will be injected into blood stream. Magnetic
field on the tumor will cause microspheres to concentrate in the tumor. Thus the release of drug
from most microspheres will take place inside the tumor. Figure 28, 29, 30 31 and 32 shows
33
some images of mice from various groups during different time interval of the experiment.
Appendix B shows in-vivo test data and images of mice during the experiment.
Tumor Size (mm3)
Variation of tumor size with time
9
8
7
6
5
4
3
2
1
0
Tumor + Particle with
drug
Tumor + Particle with
drug + Magnet
0
5
10
15
20
Days
Figure 27. Variation of tumor size at dufferent time intervals for the groups treated with
microspheres containing chemotherpeutics
Figure 28. Tumor on mouse from group 1(untreated) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 29. Tumor on mouse from group 2 (treated with pure chemotherpeutics) (a) Day 1 (b)
Day 3 (c) Day 10 (d) Day 17
34
Figure 30. Tumor on mouse from group 3 (treated with microspheres containing no drugs) (a)
Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 31. Tumor on mouse from group 4 (treated with microspheres containing drugs) (a) Day 1
(b) Day 3 (c) Day 10 (d) Day 17
Figure 32. Tumor on mouse from group 5 (treated with magnetic field and microspheres
containing drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
For the groups 4 and 5 some mice exhibited necrosis around the tumor. It has been assumed that
necrosis is caused by cyclophosphamide. Necrosis was more evident in group 5 where two mice
died due to excessive tissue damage. The slow continuous release of drug and accumulation of
drug carrier at the site of magnetic field application led to very high concentration of
cyclophosphamide in and around the tumor leading to necrosis. No necrosis was observed for
group 2 where pure drug was administered. Pure drug administration causes rapid diffusion of
drug and thus its concentration was not high enough in the tumor to cause necrosis. This shows
35
that with proper dose of drug in the carrier it is possible to obtain effective treatment. Moreover
the drug carriers were administered subcutaneously for ease of experiment. So the high
concentration of drug easily led to necrosis. Figure 33 shows necrotic site of a mouse from group
4.
Necrotic Region
Figure 33. Necrotic site on a mouse from group 4 (treated with microspheres containing drugs)
4.5 Histology Study
The hostology slides for tumors were studied. The results seemed consistent with other
experiments. It was observed that tumors from group 1 which was untreated possessed more live
cancer cells compared to the treated ones (group 2, 4 and 5). This is exhibited in figure 34, 35, 36
and 37. It was seen that tumors treated with microspheres containing drugs had less healthy
cancer cells when compared the one treated with pure chemotherapeutic. This shows that
treatment with microsphere is more effective than pure chemotherapeutic. Figure 38 shows the
tumor sections from the group treated with microspheres without drugs. There seemed to be
some dead and unhealthy cells in this case too revealing some toxicity of the microsphere itself.
36
Figure 34. Left side tumor sections of different mice from group 1 (untreated) at 10X
magnification
Figure 35. Tumor section of different mice from group 2 (treated with pure chemotherapeutic) at
10X magnification (a) Left side (b) Right side
37
Figure 36. Tumor sections of different mice from group 4 (treated with microspheres containing
drugs) at 10X magnification (a) Left side (b) Right side
Figure 37. Tumor sections of different mice from group 5 (treated with microspheres containing
drugs + magnetic field) at 10X magnification (a) Right side (b) Right side
38
Figure 38. Tumor sections of different mice from group 3 (treated with microspheres containing
no drugs) at 10X magnification (a) Left side (b) Right side
With the software Image Pro 7.0, the area of the whole tumor and the area of live cell regions in
the tumor were measured as shown in figures 39 and 40. Appendix C shows the images of
tumors and the areas considered to be containing live cells for all groups. Then percentage live
cells in each tumor was calculated using the formula given below:
(4.1)
39
Live cancer
cell regions
Figure 39. Left side tumor section of a mouse from group 1 (untreated) at 4X magnification
Live cancer
cell regions
Figure 40. Right side tumor section of a mouse from group 4 (treated with chemotherpeutics) at
4X magnification
For group 5, two mice did not survive the entire treatment period. So the tumors possessed by
them were not taken into account in the above mentioned calculation. Since the areas of live cells
calls for personal judgement, these values are approximate. Figure 41 exhibits the variation of
percentage live cancer cells in tumor for different treatments. It is evident that percentage live
40
cells for tumor treated with microsphere containing drugs is far less than the one treated with
pure chemotherapeutics only. This confirms better efficacy of treatment using these microsperes.
Percentage Area of Live Cells for different Treatments
100
90
Percentage Live Cells
80
70
60
50
40
30
20
10
0
Untreated
Pure
Chemotherapeutic
Microsphere
without Drugs
Microsphere with
Drugs
Microsphere with
Drugs + Magnetic
Field
Figure 41. Variation of percentage live cells for different treatments
The percentage live cells for tumor treated with magnetic field and microspheres containing
drugs was slighly more than the one treated with only microspheres containing drugs. This
contradicts the result obtained from tumor size measurements. This discrepancy can be
accounted by the fact that only three tumor sections for each tumor was observed and the one
that best represents all three sections was used in this calculation. The sample size for group 5
histology study was less. Also the results from tumor size measurement were very close which
was also the case for the percentage live cells.
41
CHAPTER 5
CONCLUSION AND FUTURE WORK
In this research work a novel drug delivery system was used for treatment against breast
cancer. The objective behind this work is to limit spreading of chemotherapy and achieve
targeted treatment. The targeting is achieved using magnetic field and albumin. The drug
delivery system consists of PLGA microspheres containing iron oxide magnetic nanoparticle,
albumin, fluorescent agent, chemotherapeutics (5Fu and cyclophosphamide). Magnetic
nanoparticles and albumin act as targeting agents. Albumin is attracted to cancer cells and this
causes the the microspheres to accumulate into the tumor. Application of external magnetic field
on the tumor attract magnetic nanoparticles causing microspheres to accumulate in the tumor.
Thus release of chemotherpuetic takes place inside the tumor. Also the continuous release of
drugs, at first burst release followed by slow release due to diffusion prevent rapid diffusion of
drugs away from the tumor site. This also leads to effective treatment.
ATR study on the microspheres showed that all the expected components are present.
Absorption peaks for Fe-O shows the presence of iron oxide. Peaks for P=O and C-F exhibits the
presence of both the chemotherapeutic. In-vitro test is carried out using 3T3 and also breast
cancer cells. It was observed that most of the drug is released within first three days. With
decreasing concentration of drug the effectiveness of treatment was drastically reduced. For low
conccentrations of drugs, the OD readings were similar the the control experiment which
involved only cell medium and no chemotherapeutic. The release profile of cyclophosphamide
from microspheres was studied by means of HPLC. The HPLC peak for cyclophosphamide was
around 6 to 6.5 minutes. Around 77% of drug was released within first 12 hours which was due
42
to the burst release. Then there was very slow release which was due to diffusion. From in-vivo
test results it was observed that the tumor size continued to increase in case of the group that did
not undergo any treatment and also for the group treated with microspheres containing no
chemotherapeutics. For the group treated with microspheres containing chemotherapeutics a
drastic decrease in tumor size was observed and it continued to decrease even after the treatment
was stopped.
A better control over drug release needs to be achieved in the future. It would be great if
most drug is not released during the burst release as in this case. Better manipulation of the
polymer will yield much better controlled release than the one observed in this case. The release
profile of 5 Fu from PLGA microspheres still needs to be studied. Histology study involving
organs like liver and lungs need to be studied to observe the effect of chemotherapeutics on
them. This would also help to know whether controlled release mechanism and targeted delivery
were successful in limiting diffusion of chemotherapeutics away from tumor site. Other active
targeting agents can be incorporated to achieve better targeted treatment. Aptamer specific to
breast cancer cells can be used to functionalize the drug carriers to improve the targeting
capability.
43
REFERENCES
44
REFERENCES
[1]
Pankhurst, Q. A., Connolly, J., Jones, S. K., Dobson, J., “Applications of magnetic
nanoparticles in biomedicine,” Journal of Physics D: Applied Physics, Vol. 36, 2003, pp.
167-181.
[2]
Berry, C. C., Curtis, A. S. G., “Functionalization of magnetic nanoparticles for
applications in biomedicine,” Journal of Physics D: Applied Physics, Vol. 36, 2003, pp.
198-206.
[3]
Tartaj, P., Morales, M. P., Veintemillas-Verdaguer, S., González-Carreño, T., Serna, C.
J., “The preparation of magnetic nanoparticles for applications in biomedicine,” Journal
of Physics D: Applied Physics, Vol. 36, 2006, pp. 182-197.
[4]
Pankhurst, Q. A., Thanh, N. K. T., Jones, S. K., Dobson, J., “Progress in applications of
magnetic nanoparticles in biomedicine,” Journal of Physics D: Applied Physics, Vol. 42,
2009, pp. 1-15.
[5]
Takeda, S., Mishima, F., Fujimoto, S., Izumi, Y., Nishijima, S., “Development of
magnetically targeted drug delivery using superconducting magnet,” Journal of
Magnetism and Magnetic Materials, Vol. 311, 2007, pp. 367-371.
Ciofani, G., Raffa, V., Obata, Y., Menciassi, A., Dario, P., Takeoka, S., “Magnetic driven
alginate nanoparticles for targeted drug delivery,” Current Nanoscience, Vol. 4, 2008, pp.
212-218.
[6]
[7]
Mu, B., Liu, P., Du, P., Dong, Y., Lu, C., “Magnetic-targeted pH responsive drug
delivery system via layer-by-layer self-assembly of polyelectrolytes onto drug –
containing emulsion droplets and its controlled release,” Journal of Polymer Science Part
A: Polymer Chemistry, Vol. 49, 2011, pp. 1969-1976.
[8]
Goodrazi, A., Sahoo, Y., Swihart, M. T., Prasad, P. N., “Aqueous ferrofluid of citric acid
coated magnetite particles,” Materials Research Society Symposium Proceedings, Vol.
789, Materials Research Society, 2004.
[9]
Cheng, J., Teply, B. A., Jeong, S. Y., Yim, C. H., Ho, D., Sherifi, I., Jon, S., Farokhzad,
O. C., khademhosseini, A., Langer, R. S., “Magnetically responsive polymeric
microparticles for oral delivery of protein drugs,” Pharmaceutical Research, Vol. 23, No.
3, 2006, pp. 557-564.
[10]
Banerjee, S. S., Chen, D., “A multifunctional magnetic nanocarrier bearing fluorescent
dye for targeted drug delivery by enhanced two-photon triggered release,”
Nanotechnology, Vol. 20, 2009, pp. 1-9.
Rutnakornpituk, M., Meerod, S., Boontha, B., Wichai, U., “Magnetic core-bilayer shell
nanoparticle: A novel vehicle for entrapment of poorly water-soluble drugs,” Polymer,
Vol. 50, 2009, pp. 3508-3515.
[11]
45
[12]
Asmatulu, R., Fakhari, A., Wamocha, H. L., Chu, H. Y., Chen, Y. Y., Eltabey, M. M.,
Hamdeh, H. H., Ho, J. C., “Drug carrying magnetic nanocomposite particles for potential
drug delivery systems,” Journal of Nanotechnology, 2009, pp. 1-6.
[13]
Asmatulu, R., Zalich, M. A., Claus, R. O., Riffle, J. S., “Synthesis, characterization and
targeting of biodegradable magnetic nanocomposite particles by external magnetic
fields,” Journal of Magnetism and Magnetic Materials, Vol. 292, 2005, pp. 108-119.
[14]
Oh, W. K., Yoon, H., Jang, J., “Size control magnetic carbon nanoparticles for drug
delivery,” Biomaterials, Vol. 31, 2010, pp. 1342-1348.
[15]
Avilés, M. O., Ebner, A. D., Ritter, J. A., “In vitro study of magnetic particle seeding for
implant-assisted-magnetic drug targeting: Seed and magnetic drug carrier particle
capture,” Journal of Magnetism and Magnetic Materials, Vol. 321, 2009, pp. 1586-1590.
[16]
Fernández-Pacheco, R., Marquina, C., Valdivia, J. G., Gutiérrez, M., Romero, M. S.,
Cornudella, R., Laborda, A., Viloria, A., Higuera, T., García, A., Jalón, J. A. G., Ibarra,
M. R., “Magnetic nanoparticles for local drug delivery using magnetic implants,” Journal
of Magnetism and Magnetic Materials, Vol. 311, 2007, pp. 318-322.
[17]
Wilson, M. W., Kerlan, R. K., Fidelman, N. A., Venook, A. P., Laberge, J. M., Koda, J.,
Gordon, R. L., “Hepatocellular carcinoma: regional therapy with a magnetic targeted
carrier bound to doxorubicin in a dual MR imaging/conventional angiography suiteinitial experience with four patients,” Radiology, Vol. 230, 2004, pp. 287-293.
[18]
Saravanan, M., Anbu, J., Maharanjan, G., Pillai, K, S., “Targeted delivery of diclofenac
sodium via gelatin magnetic microspheres formulated for intra-arterial administration,”
Journal of Drug Targeting, Vol. 16, No. 5, 2008, pp. 366-378.
[19]
Bao, T., Hiep, N., Kim, Y., Yang, H., Lee, B., “Fabrication and characterization of
porous poly(lactic-co-glycolic acid) (PLGA) microspheres for use as a drug delivery
system,” Journal of Materials Science, Vol. 26, 2011, pp. 2510-2517.
[20]
Lin, R., Ng, L. S., Wang, C., “In vitro study of anticancer drug doxorubicin in PLGAbased microparticles,” Biomaterials, Vol. 26, 2005, pp. 4476-4485.
[21]
Wei, G., Pettway, G. J., McCauley, L. K., Ma, P. X., “The release profiles and bioactivity
of parathyroid hormone from poly(lactic-co-glycolic acid) microspheres,” Biomaterials,
Vol. 25, 2004, pp. 345-352.
[22]
Kratz, F., “Albumin as a drug carrier: design of prodrugs, drug conjugates and
nanoparticles,” Journal of controlled release, Vol. 132, 2008, pp. 171-183.
[23]
Stehle, G., Sinn, H., Wunder, A., Schrenk, H. H., Stewart, J. C. M., Hartung, G., MaierBorst, W., Heene, D. L., “Plasma protein (albumin) catabolism by the tumor itself –
46
implications for tumor metabolism and the genesis of cachexia,” Critical Reviews in
Oncology/Hematology, Vol. 26, No. 2, 1997, pp. 77-100.
[24]
Wunder, A., Müller-Ladner, U., Stelzer, E. H. K., Funk, J., Neumann, E., Stehle, G., Pap,
T., Sinn, H., Gay, S., Fiehn, C., “Albumin-based drug delivery as novel therapeutic
approach for rheumatoid arthritis,” The Journal of Immunology, Vol. 170, No. 9, 2003,
pp. 4793-4801.
[25]
Agarwal, A., Majumder, S., Agrawal, H., Majumdar, S., Agrawal, G. P., “Cationized
albumin conjugated solid lipid nanoparticles as vectors for brain delivery of an anticancer drug,” Current Nanoscience, Vol. 7, 2011, pp. 71-80.
[26]
Nuemann, E., Frei, E., Funk, D., Becker, M. D., Schrenk, H., Müller-Ladner, U., Fiehn,
C., “Native albumin for targeted drug delivery,” Expert Opinion on Drug Delivery, Vol.
7, No. 8, 2010, pp. 915-925.
[27]
Alexis, F., Basto, P., Levy-Nissenbaum, E., Radovic-Moreno, A. F., Zhang, L., pridgen,
E., Wang, A. Z., Marein, S. L., Westerhof, K., “HER-2-targeted nanoparticle-affibody
bioconjugates for cancer therapy,” ChemMedChem, Vol. 3, 2008, pp. 1839-1843.
[28]
Cao, Z., Tong, R., Mishra, A., Xu, W., Wong, G. C. L., Cheng, J., Lu, Y., “Reversible
cell-specific drug delivery with aptamer-funtionalized liposomes,” Angewandte Chemie
International Edition, Vol. 48, 2009, pp. 6494-6498.
[29]
Huang, Y., Lin, Y., Lin, Z., Chang, H., “Aptamer-modified gold nanoparticles for
targeting breast cancer cells through light scattering,” Journal of Nanoparticle Research,
Vol. 11, No. 4, 2009, pp. 775-783.
[30]
Kensler, T. T., Behme, R. J., Brooke, D., “High-performance liquid chromatography
analysis of cyclophosphamide,” Journal of Pharmaceutical Sciences, Vol. 68, No. 2,
1979, pp. 172-174.
[31]
Tomayko, M. M., Reynolds, C. P., “Dtermination of subcutaneous tumor size in athymic
(nude) mice,” Cancer Chemother Pharmacol, Vol. 24, 1989, pp. 148-154.
[32]
Mohapatra, S. D., “FTIR analysis of Bi2O3-B2O3-Fe2O3 glass system doped with Nd2O3,”
Thesis, Department of Ceramic Engineering, National Institute of Technology, Rourkela,
India, 2009.
[33]
Infrared Spectra, URL: http://www.mpcfaculty.net/ron_rinehart/12A/IR/primclue.htm
[cited 5 March 2011]
[34]
Bobkova, N. M., Zayants, N. I., Glasova, M. P., “IR spectra of calcium phosphate-silicate
glasses as the basis of biopyrocerams,” Journal of Applied Spectroscopy, Vol. 61, 1994,
pp. 637-639.
47
APPENDICES
48
APPENDIX A
HPLC PEAKS AND CALCULATIONS OF RELEASE RATE OF CYCLOPHOSPHAMIDE
Figure 42. HPLC peak for solution of 10 mg/ml of cyclophosphamide in PBS for first run
Figure 43. HPLC peak for solution of 10 mg/ml of cyclophosphamide in PBS for second run
49
Figure 44. HPLC peak for solution of 5 mg/ml of cyclophosphamide in PBS for first run
Figure 45. HPLC peak for solution of 5 mg/ml of cyclophosphamide in PBS for second run
50
Figure 46. HPLC peak for solution of 2.5 mg/ml of cyclophosphamide in PBS for first run
Figure 47. HPLC peak for solution of 2.5 mg/ml of cyclophosphamide in PBS for second run
51
Figure 48. HPLC peak for solution of 1.25 mg/ml of cyclophosphamide in PBS for first run
Figure 49. HPLC peak for solution of 1.25 mg/ml of cyclophosphamide in PBS for second run
52
Figure 50. HPLC peak for solution of 0.625 mg/ml of cyclophosphamide in PBS for first run
Figure 51. HPLC peak for solution of 0.625 mg/ml of cyclophosphamide in PBS for second run
53
Figure 52. HPLC peak for 1 hour sample for first run
Figure 53. HPLC peak for 1 hour sample for second run
54
Figure 54. HPLC peak for 2 hour sample for first run
Figure 55. HPLC peak for 2 hour sample for second run
55
Figure 56. HPLC peak for 4 hour sample for first run
Figure 57. HPLC peak for 4 hour sample for second run
56
Figure 58. HPLC peak for 8 hour sample for first run
Figure 59. HPLC peak for 8 hour sample for second run
57
Figure 60. HPLC peak for 12 hour sample for first run
Figure 61. HPLC peak for 12 hour sample for second run
58
Figure 62. HPLC peak for 24 hour sample for first run
Figure 63. HPLC peak for 24 hour sample for second run
59
Figure 64. HPLC peak for 72 hour sample for first run
Figure 65. HPLC peak for 72 hour sample for second run
60
Figure 66. HPLC peak for 10 mg/ml cyclophosphamide in acetonitrile for first run
Figure 67. HPLC peak for 10 mg/ml cyclophosphamide in acetonitrile for second run
61
Figure 68. HPLC peak for 5 mg/ml cyclophosphamide in acetonitrile for first run
Figure 69. HPLC peak for 5 mg/ml cyclophosphamide in acetonitrile for second run
62
Figure 70. HPLC peak for 2.5 mg/ml cyclophosphamide in acetonitrile for first run
Figure 71. HPLC peak for 2.5 mg/ml cyclophosphamide in acetonitrile for second run
63
Figure 72. HPLC peak for 1.25 mg/ml cyclophosphamide in acetonitrile for first run
Figure 73. HPLC peak for 1.25 mg/ml cyclophosphamide in acetonitrile for second run
64
Figure 74. HPLC peak for 0.625 mg/ml cyclophosphamide in acetonitrile for first run
Figure 75. HPLC peak for 0.625 mg/ml cyclophosphamide in acetonitrile for second run
65
Figure 76. HPLC peak of cyclophosphamide for 100 mg/ml drug carrier in acetonitrile for first
run
Figure 77. HPLC peak of cyclophosphamide for 100 mg/ml drug carrier in acetonitrile for
second run
66
TABLE 1
DATA FOR CALIBRATION CHART OF CYCLOPHOSPHAMIDE IN PBS
Concentration
(mg/ml)
10
5
2.5
1.25
0.625
Area 1
(mVolts*sec)
2759
1738
977
448
192
Area 2
(mVolts*sec)
2906
1754
850
436
177
Average Area
(mVolts*sec)
2832.5
1746
913.5
442
184.5
STD
103.9446968
11.3137085
89.80256121
8.485281374
10.60660172
TABLE 2
DATA FOR CONCENTRATION OF CYCLOPHOSPHAMIDE IN THE PBS SAMPLE
Time (hours)
1
2
4
8
12
24
72
Area 1
(mVolts*sec)
303
239
178
147
110
85.7
71.3
Concentration 1
(mg/ml)
0.9999
0.7887
0.5874
0.4851
0.363
0.28281
0.23529
Area 2
(mVolts*sec)
285
233
172
140
113
83.3
71.2
Concentration 2
(mg/ml)
0.9405
0.7689
0.5676
0.462
0.3729
0.27489
0.23496
Calculation for determining the cumulative drug concentration data for release profile:
Relation between area and concentration for cyclophosphamide in PBS:
Based on above formula the followings are calculated:
Calculation for 1 hour sample:
Run 1: Concentration of drug in 1 hour sample:
Run 2: Concentration of drug in 1 hour sample:
Calculation for 2 hour sample:
Run 1: Amount of drug in 30 µl of the first hour sample that is taken out =
= 0.029997 mg
67
Amount of drug in 100 µl of 2 hour sample =
= 0.07887 mg
Total amount of drug that should be in 100 µl of 2 hour sample = 0.029997 + 0.07887
= 0.108867 mg
Actual concentration for 2 hour sample = 0.108867/0.1=1.08867 mg/ml
Run 2: Amount of drug in 30 µl of the first hour sample that is taken out =
= 0.028215 mg
Amount of drug in 100 µl of 2 hour sample =
= 0.07689 mg
Total amount of drug that should be in 100 µl of 2 hour sample = 0.028215 + 0.07689
= 0.105105 mg
Actual concentration for 2 hour sample = 0.105105/0.1=1.05105 mg/ml
Calculation for 4 hour sample:
Run 1: Amount of drug in 30 µl of the first hour sample that is taken out = 0.029997 mg
Amount of drug in 30 µl of the 2 hour sample that is taken out =
= 0.023661 mg
Amount of drug in 100 µl of 4 hour sample =
= 0.05874 mg
Total amount of drug that should be present in 100 µl of 4 hour sample:
0.029997 + 0.023661+ 0.05874
= 0.112398 mg
Actual concentration of 4 hour sample = 0.112398/0.1
= 1.12398 mg/ml
Run 2: Amount of drug in 30 µl of the first hour sample that is taken out = 0.028215 mg
68
Amount of drug in 30 µl of the 2 hour sample that is taken out =
= 0.023067 mg
Amount of drug in 100 µl of 4 hour sample =
= 0.05676 mg
Total amount of drug that should be present in 100 µl of 4 hour sample:
0.028215 + 0.023067 + 0.05676
= 0.108042 mg
Actual concentration of 4 hour sample = 0.108042 /0.1
= 1.08042 mg/ml
Calculation for 8 hour sample:
Run 1: Amount of drug in 30 µl of the first hour sample that is taken out = 0.029997 mg
Amount of drug in 30 µl of the 2 hour sample that is taken out = 0.023661 mg
Amount of drug in 30 µl of the 4 hour sample that is taken out =
= 0.017622 mg
Amount of drug in 100 µl of 8 hour sample =
= 0.04851 mg
Total amount of drug that should be present in 100 µl of 8 hour sample:
0.029997 + 0.023661 + 0.017622 + 0.04851
= 0.11979 mg
Actual concentration of 8 hour sample = 0.11979/0.1
= 1.1979 mg/ml
Run 2: Amount of drug in 30 µl of the first hour sample that is taken out = 0.028215 mg
Amount of drug in 30 µl of the 2 hour sample that is taken out = 0.023067 mg
Amount of drug in 30 µl of the 4 hour sample that is taken out =
69
= 0.017028 mg
Amount of drug in 100 µl of 8 hour sample =
= 0.0462 mg
Total amount of drug that should be present in 100 µl of 8 hour sample:
0.028215 + 0.023067 + 0.017028 + 0.0462
= 0.11451 mg
Actual concentration of 8 hour sample = 0.11451/0.1
= 1.1451 mg/ml
Calculation for 12 hour sample:
Run 1: Amount of drug in 30 µl of the first hour sample that is taken out = 0.029997 mg
Amount of drug in 30 µl of the 2 hour sample that is taken out = 0.023661 mg
Amount of drug in 30 µl of the 4 hour sample that is taken out = 0.017622 mg
Amount of drug in 30 µl of the 8 hour sample that is taken out =
= 0.014553 mg
Amount of drug in 100 µl of 12 hour sample =
= 0.0363 mg
Total amount of drug that should be present in 100 µl of 12 hour sample:
0.029997 + 0.023661 + 0.017622 + 0.014553 + 0.0363
= 0.122133 mg
Actual concentration of 12 hour sample = 0.122133/0.1
= 1.22133 mg/ml
Run 2: Amount of drug in 30 µl of the first hour sample that is taken out = 0.028215 mg
Amount of drug in 30 µl of the 2 hour sample that is taken out = 0.023067 mg
Amount of drug in 30 µl of the 4 hour sample that is taken out = 0.017028 mg
70
Amount of drug in 30 µl of the 8 hour sample that is taken out =
= 0.01386 mg
Amount of drug in 100 µl of 12 hour sample =
= 0.03729 mg
Total amount of drug that should be present in 100 µl of 12 hour sample:
0.028215 + 0.023067 + 0.017028 + 0.01386 + 0.03729
= 0.11946 mg
Actual concentration of 12 hour sample = 0.11946 /0.1
= 1.1946 mg/ml
Calculation for 24 hour sample:
Run 1: Amount of drug in 30 µl of the first hour sample that is taken out = 0.029997 mg
Amount of drug in 30 µl of the 2 hour sample that is taken out = 0.023661 mg
Amount of drug in 30 µl of the 4 hour sample that is taken out = 0.017622 mg
Amount of drug in 30 µl of the 8 hour sample that is taken out = 0.014553 mg
Amount of drug in 30 µl of the 12 hour sample that is taken out =
= 0.01089 mg
Amount of drug in 100 µl of 24 hour sample =
= 0.028281 mg
Total amount of drug that should be present in 100 µl of 24 hour sample:
0.029997 + 0.023661 + 0.017622 + 0.014553 + 0.01089 + 0.028281
=0.125004 mg
Actual concentration of 12 hour sample = 0.125004/0.1
= 1.25004 mg/ml
Run 2: Amount of drug in 30 µl of the first hour sample that is taken out = 0.028215 mg
71
Amount of drug in 30 µl of the 2 hour sample that is taken out = 0.023067 mg
Amount of drug in 30 µl of the 4 hour sample that is taken out = 0.017028 mg
Amount of drug in 30 µl of the 8 hour sample that is taken out = 0.01386 mg
Amount of drug in 30 µl of the 12 hour sample that is taken out =
= 0.011187 mg
Amount of drug in 100 µl of 24 hour sample =
= 0.027489 mg
Total amount of drug that should be present in 100 µl of 24 hour sample:
0.028215 + 0.023067 + 0.017028 + 0.01386 + 0.011187 + 0.027489
= 0.120846 mg
Actual concentration of 12 hour sample = 0.120846/0.1
= 1.20846 mg/ml
Calculation for 72 hour sample:
Run 1: Amount of drug in 30 µl of the first hour sample that is taken out = 0.029997 mg
Amount of drug in 30 µl of the 2 hour sample that is taken out = 0.023661 mg
Amount of drug in 30 µl of the 4 hour sample that is taken out = 0.017622 mg
Amount of drug in 30 µl of the 8 hour sample that is taken out = 0.014553 mg
Amount of drug in 30 µl of the 12 hour sample that is taken out = 0.01089 mg
Amount of drug in 30 µl of the 24 hour sample that is taken out =
= 0.0084843 mg
Amount of drug in 100 µl of 72 hour sample =
= 0.023529 mg
Total amount of drug that should be present in 100 µl of 72 hour sample:
0.029997 + 0.023661 + 0.017622 + 0.014553 + 0.01089 + 0.0084843 + 0.023529
72
=0.1287363 mg
Actual concentration of 12 hour sample = 0.1287363/0.1
= 1.287363 mg/ml
Run 2: Amount of drug in 30 µl of the first hour sample that is taken out = 0.028215 mg
Amount of drug in 30 µl of the 2 hour sample that is taken out = 0.023067 mg
Amount of drug in 30 µl of the 4 hour sample that is taken out = 0.017028 mg
Amount of drug in 30 µl of the 8 hour sample that is taken out = 0.01386 mg
Amount of drug in 30 µl of the 12 hour sample that is taken out = 0.011187 mg
Amount of drug in 30 µl of the 24 hour sample that is taken out =
= 0.0082467 mg
Amount of drug in 100 µl of 72 hour sample =
= 0.023496 mg
Total amount of drug that should be present in 100 µl of 72 hour sample:
0.028215 + 0.023067 + 0.017028 + 0.01386 + 0.011187 + 0.0082467 + 0.023496
=0.1250997 mg
Actual concentration of 12 hour sample = 0.1250997 /0.1
= 1.250997 mg/ml
TABLE 3
CUMULATIVE CONCENTRATION OF CYCLOPHOSPHAMIDE IN PBS SAMPLE
Time (hours)
0
1
2
4
8
12
Cumulative Concentration
(mg/ml)
0
0.9999
1.08867
1.12398
1.1979
1.22133
0
0.9405
1.05105
1.08042
1.1451
1.1946
73
Average
Concentration
(mg/ml)
0
0.9702
1.06986
1.1022
1.1715
1.207965
STD
0
0.042002143
0.026601357
0.030801571
0.037335238
0.018900964
TABLE 3 (continued)
Time (hours)
Cumulative Concentration
(mg/ml)
24
72
1.25004
1.287363
1.20846
1.250997
Average
Concentration
(mg/ml)
1.22925
1.26918
STD
0.0294015
0.025714645
TABLE 4
DATA FOR CALIBRATION CHART OF CYCLOPHOSPHAMIDE IN ACETONITRILE
Concentration
(mg/ml)
10
5
2.5
1.25
0.625
Area 1
(mVolts*sec)
3517
2076
1349
787
464
Area 2
(mVolts*sec)
3846
1794
1298
773
434
Average Area
(mVolts*sec)
3681.5
1935
1323.5
780
449
STD
232.638131
199.4041123
36.06244584
9.899494937
21.21320344
TABLE 5
DATA FOR CYCLOPHOSPHAMIDE FROM 100MG/ML SUSPENSION OF DRUG
CARRIER IN ACETONITRILE
Area 1
(mVolts*sec)
627
Concentration 1
(mg/ml)
1.635879775
Area 2
(mVolts*sec)
574
Formula for percentage release of cyclophosphamide:
74
Concentration
(mg/ml)
1.497599666
TABLE 6
DATA FOR CUMULATIVE PERCENTAGE RELEASE OF CYCLOPHOSPHAMMIDE IN
PBS
Time
(hours)
0
1
2
4
8
12
24
72
Concentration Percentage Concentration Percentage Average
1 (mg/ml)
Release
2 (mg/ml)
Release
Percentage
Release
0
0
0
0
0
0.9999
61.123
0.9405
62.800
61.962
1.08867
66.550
1.05105
70.182
68.366
1.12398
68.708
1.08042
72.143
70.426
1.1979
73.227
1.1451
76.462
74.845
1.22133
74.659
1.1946
79.768
77.213
1.25004
76.414
1.20846
80.693
78.553
1.287363
78.695
1.250997
83.533
81.114
75
STD
0
1.186116
2.568775
2.429239
2.287988
3.612422
3.02585
3.420996
APPENDIX B
IMAGES OF TUMORS FROM IN-VIVO TEST
Figure 78. Left side tumor on mouse (723) from group 1(untreated) (a) Day 1 (b) Day 3 (c) Day
10 (d) Day 17
Figure 79. Right side tumor on mouse (723) from group 1(untreated) (a) Day 1 (b) Day 3 (c) Day
10 (d) Day 17
Figure 80. Left side tumor on mouse (724) from group 1(untreated) (a) Day 1 (b) Day 3 (c) Day
10 (d) Day 17
76
APPENDIX B (continued)
Figure 81. Right side tumor on mouse (724) from group 1(untreated) (a) Day 1 (b) Day 3 (c) Day
10 (d) Day 17
Figure 82. Left side tumor on mouse (725) from group 1(untreated) (a) Day 1 (b) Day 3
Figure 83. Right side tumor on mouse (724) from group 1(untreated) (a) Day 1 (b) Day 3
77
APPENDIX B (continued)
Figure 84. Left side tumor on mouse (726) from group 1(untreated) (a) Day 1 (b) Day 3 (c) Day
10 (d) Day 17
Figure 85. Right side tumor on mouse (726) from group 1(untreated) (a) Day 1 (b) Day 3 (c) Day
10 (d) Day 17
Figure 86. Left side tumor on mouse (727) from group 2 (treated with pure chemotherpeutics) (a)
Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 87. Right side tumor on mouse (727) from group 2 (treated with pure chemotherpeutics)
(a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
78
APPENDIX B (continued)
Figure 88. Left side tumor on mouse (728) from group 2 (treated with pure chemotherpeutics) (a)
Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 89. Right side tumor on mouse (728) from group 2 (treated with pure chemotherpeutics)
(a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 90. Left side tumor on mouse (729) from group 2 (treated with pure chemotherpeutics) (a)
Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 91. Right side tumor on mouse (729) from group 2 (treated with pure chemotherpeutics)
(a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
79
APPENDIX B (continued)
Figure 92. Left side tumor on mouse (730) from group 2 (treated with pure chemotherpeutics) (a)
Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 93. Right side tumor on mouse (730) from group 2 (treated with pure chemotherpeutics)
(a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 94. Left side tumor on mouse (731) from group 3 (treated with microsphere without
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 95. Right side tumor on mouse (731) from group 3 (treated with microsphere without
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
80
APPENDIX B (continued)
Figure 96. Left side tumor on mouse (732) from group 3 (treated with microsphere without
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 97. Right side tumor on mouse (732) from group 3 (treated with microsphere without
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 98. Left side tumor on mouse (733) from group 3 (treated with microsphere without
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 99. Right side tumor on mouse (733) from group 3 (treated with microsphere without
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
81
APPENDIX B (continued)
Figure 100. Left side tumor on mouse (734) from group 3 (treated with microsphere without
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 101. Right side tumor on mouse (734) from group 3 (treated with microsphere without
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 102. Left side tumor on mouse (735) from group 4 (treated with microsphere containing
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 103. Right side tumor on mouse (735) from group 4 (treated with microsphere containing
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
82
APPENDIX B (continued)
Figure 104. Left side tumor on mouse (736) from group 4 (treated with microsphere containing
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 105. Right side tumor on mouse (736) from group 4 (treated with microsphere containing
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 106. Left side tumor on mouse (737) from group 4 (treated with microsphere containing
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 107. Right side tumor on mouse (737) from group 4 (treated with microsphere containing
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
83
APPENDIX B (continued)
Figure 108. Left side tumor on mouse (738) from group 4 (treated with microsphere containing
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 109. Right side tumor on mouse (738) from group 4 (treated with microsphere containing
drugs) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 110. Left side tumor on mouse (739) from group 5 (treated with microsphere containing
drugs + magnetic field) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 111. Right side tumor on mouse (739) from group 5 (treated with microsphere containing
drugs + magnetic field) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
84
APPENDIX B (continued)
Figure 112. Left side tumor on mouse (740) from group 5 (treated with microsphere containing
drugs + magnetic field) (a) Day 1 (b) Day 3
Figure 113. Right side tumor on mouse (740) from group 5 (treated with microsphere containing
drugs + magnetic field) (a) Day 1 (b) Day 3
Figure 114. Left side tumor on mouse (741) from group 5 (treated with microsphere containing
drugs + magnetic field) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
85
APPENDIX B (continued)
Figure 115. Right side tumor on mouse (741) from group 5 (treated with microsphere containing
drugs + magnetic field) (a) Day 1 (b) Day 3 (c) Day 10 (d) Day 17
Figure 116. Left side tumor on mouse (742) from group 5 (treated with microsphere containing
drugs + magnetic field) (a) Day 1 (b) Day 3
Figure 117. Right side tumor on mouse (742) from group 5 (treated with microsphere containing
drugs + magnetic field) (a) Day 1 (b) Day 3
86
TABLE 7
DATA FOR TUMOR SIZE AT VARIOUS TIME INTERVALS
No.
of
days
Type
of
treat
ment
1
3
6
8
10
13
15
17
Volume of tumor (mm3)
Untre
ated
Pur
e
dru
gs
Micros
phere
without
drugs
Micros
phere
with
drugs
6.610
4
10.57
02
17.23
51
21.81
66
26.09
27
44.85
5
52.88
35
61.52
29
7.2
322
5.4
323
5.0
396
3.7
961
5.9
232
4.7
778
6.8
068
9.2
939
6.7495
8.2794
6.8395
4.7124
10.864
7
13.220
9
14.791
7
23.529
2
35.081
1
44.178
6
2.6834
1.6690
0.8181
0.8399
0.7418
0.7106
Standard deviation
Micros Untre
phere
ated
with
drugs +
magnet
ic field
5.3096 1.299
6
4.6919 1.238
1
2.4871 3.641
5
1.6362 3.559
4
0.6218 6.081
7
0.4581 13.50
98
0.3927 14.87
98
0.1963 20.95
43
87
Pur
e
dru
gs
Micros
phere
without
drugs
Micros
phere
with
drugs
1.5
265
1.4
883
1.4
617
1.0
405
2.3
600
1.1
800
1.4
696
2.2
258
1.8218
2.4842
Micros
phere
with
drugs +
magnet
ic field
1.2390
2.1225
1.3505
0.9337
3.0872
0.6558
0.7252
4.1455
0.4360
0.4122
4.5484
0.1536
0.3772
8.3589
0.3005
0.2581
9.8031
0.1993
0.2581
13.637
4
0.2112
0.0756
TABLE 8
DATA FOR IN-VITRO TEST WITH 3T3 CELLS
Concentr
ation
(mg/ml)
Day 1
release
Average
Day 3
release
Average
Day 5
release
Average
Medium
Average
10
5
2.5
1.2
5
0.0
5
0.0
60
0.0
55
0.0
45
0.0
48
0.0
47
0.4
79
0.3
89
0.4
34
1.1
40
1.1
84
1.1
62
0.0
43
0.0
43
0.0
43
0.0
48
0.0
66
0.0
57
0.5
31
0.4
05
0.4
68
1.0
07
1.2
64
1.1
35
0.0
41
0.0
53
0.0
47
0.0
73
0.0
90
0.0
82
0.6
52
0.5
78
0.6
15
1.0
36
1.1
37
1.0
87
0.0
45
0.0
42
0.0
44
0.3
02
0.2
46
0.2
74
0.7
36
0.6
23
0.6
80
0.9
68
1.1
12
1.0
40
0.6
25
0.0
44
0.0
47
0.0
46
0.5
02
0.5
41
0.5
21
0.8
43
0.7
00
0.7
71
1.0
55
1.1
55
1.1
05
0.31
25
0.04
2
0.04
3
0.04
3
0.60
6
0.65
4
0.63
0
0.77
9
0.86
7
0.82
3
0.91
0
1.08
2
0.10
0
0.15
625
0.078
125
0.039
063
0.019
531
0.009
766
0.004
883
0.071
0.084
0.198
0.644
0.778
0.874
0.061
0.055
0.387
0.569
0.743
0.805
0.066
0.069
0.293
0.606
0.760
0.840
0.697
0.756
0.851
1.026
1.048
1.137
0.691
0.696
0.764
0.949
0.921
1.069
0.694
0.726
0.807
0.987
0.984
1.103
0.905
1.159
1.019
1.026
1.156
0.975
1.013
1.010
0.935
0.988
1.147
1.167
0.959
1.084
0.977
1.007
1.152
1.071
1.163
1.141
1.051
1.121
1.124
1.013
1.069
1.104
1.071
1.099
1.016
0.988
1.116
1.123
1.061
1.110
1.070
1.000
88
TABLE 9
DATA FOR IN-VITRO TEST WITH BREAST CANCER CELLS
Concentra
tion
(mg/ml)
Day 1
release
Average
Day 3
release
Average
Day 5
release
Average
Day 7
release
Average
Medium
Average
10
5
0.0
74
0.0
86
0.0
80
0.0
73
0.0
87
0.0
80
0.9
25
0.8
36
0.8
81
1.5
59
1.5
19
1.5
39
1.6
99
2.1
14
1.9
07
0.0
88
0.0
86
0.0
87
0.0
96
0.0
97
0.0
96
1.0
86
1.1
63
1.1
24
1.6
72
1.5
93
1.6
32
2.1
14
1.5
58
1.8
36
2.5
0.0
78
0.0
82
0.0
80
0.1
87
0.1
98
0.1
92
1.5
44
1.5
23
1.5
34
1.7
50
1.6
01
1.6
75
2.0
55
2.0
44
2.0
49
1.2
5
0.0
86
0.0
81
0.0
83
0.4
52
0.4
69
0.4
61
1.5
87
1.5
19
1.5
53
1.6
06
1.6
07
1.6
07
2.2
27
1.1
78
1.7
02
0.6
25
0.08
5
0.08
8
0.08
6
0.72
6
0.64
2
0.68
4
1.69
7
1.66
4
1.68
0
1.71
3
1.56
1
1.63
7
2.10
3
2.20
8
2.15
6
0.31
25
0.156
25
0.0781
25
0.0390
63
0.0195
31
0.0097
66
0.055
0.121
0.358
0.621
1.151
1.408
0.108
0.124
0.342
0.945
1.180
1.696
0.081
0.122
0.350
0.783
1.165
1.552
0.923
1.071
1.866
1.591
1.421
1.942
0.827
0.915
1.387
2.330
1.829
1.935
0.875
0.993
1.626
1.961
1.625
1.938
1.756
1.511
1.546
1.192
1.277
1.196
1.506
1.697
1.750
1.675
1.830
1.508
1.631
1.604
1.648
1.433
1.554
1.352
1.447
1.934
1.755
1.643
3.059
1.708
1.506
1.314
1.621
1.666
1.485
1.632
1.477
1.624
1.688
1.654
2.272
1.670
1.759
1.989
1.845
2.080
2.357
1.732
1.877
2.212
2.385
3.038
2.273
2.538
1.818
2.101
2.115
2.559
2.315
2.135
89
APPENDIX C
HISTOLOGY IMAGES OF TUMORS
Figure 118. Tumor sections at 4X magnification of the same mouse (723) from group
1(untreated) (a) Left side (b) Right side
Figure 119. Tumor sections at 4X magnification of the same mouse (724) from group
1(untreated) (a) Left side (b) Right side
90
APPENDIX C (continued)
Figure 120. Tumor sections at 4X magnification of the same mouse (726) from group
1(untreated) (a) Left side (b) Right side
Figure 121. Tumor sections at 4X magnification of the same mouse (727) from group 2(treated
with pure chemotherapeutics) (a) Left side (b) Right side
91
APPENDIX C (continued)
Figure 122. Tumor sections at 4X magnification of the same mouse (728) from group 2(treated
with pure chemotherapeutics) (a) Left side (b) Right side
Figure 123. Tumor sections at 4X magnification of the same mouse (729) from group 2(treated
with pure chemotherapeutics) (a) Left side (b) Right side
92
APPENDIX C (continued)
Figure 124. Tumor sections at 4X magnification of the same mouse (730) from group 2(treated
with pure chemotherapeutics) (a) Left side (b) Right side
Figure 125. Tumor sections at 4X magnification of the same mouse (731) from group 3(treated
with microspheres containing no drugs) (a) Left side (b) Right side
93
APPENDIX C (continued)
Figure 126. Tumor sections at 4X magnification of the same mouse (732) from group 3(treated
with microspheres containing no drugs) (a) Left side (b) Right side
Figure 127. Tumor sections at 4X magnification of the same mouse (733) from group 3(treated
with microspheres containing no drugs) (a) Left side (b) Right side
94
APPENDIX C (continued)
Figure 128. Tumor sections at 4X magnification of the same mouse (734) from group 3(treated
with microspheres containing no drugs) (a) Left side (b) Right side
Figure 129. Tumor sections at 4X magnification of the same mouse (735) from group 4(treated
with microspheres containing drugs) (a) Left side (b) Right side
95
APPENDIX C (continued)
Figure 130. Tumor sections at 4X magnification of the same mouse (736) from group 4(treated
with microspheres containing drugs) (a) Left side (b) Right side
Figure 131. Tumor sections at 4X magnification of the same mouse (737) from group 4(treated
with microspheres containing drugs) (a) Left side (b) Right side
96
APPENDIX C (continued)
Figure132. Right side tumor section at 4X magnification of the same mouse (738) from group
4(treated with microspheres containing drugs)
Figure 133. Right side tumor section at 4X magnification of the same mouse (739) from group
5(treated with magnetic field and microspheres containing drugs)
97
APPENDIX C (continued)
Figure 134. Right side tumor section at 4X magnification of the same mouse (741) from group
5(treated with magnetic field and microspheres containing drugs)
TABLE 10
DATA FOR PERCENTAGE LIVE CANCER CELLS IN TUMORS
Treatment Type
Untreated
Pure
Chemotherapeutics
Microspheres without
Chemotherapeutics
Total
Tumor
Area (µm2)
2940198
3515143
3502046
3517055
3347443.7
3502675
2703230
2356861
2639058
3093948
1476795
2965149
3398283
2959196
1234708
734062.8
3419295
3235183
3456158
Area of Live
Cancer Cells
(µm2)
2528280
2956097
3421721
2886227
3347443.7
2583129.5
737430.3371
651252.4756
595458.9267
584961.9375
548146.3945
1006960.665
2221409.715
785470.397
741873.7877
485972.4424
1884312.459
1732575.728
973617.4617
98
Percentage
Live Cells
85.9901
84.0961
97.7063
82.0637
100
73.7473
27.2796
27.6322
22.5633
18.9067
37.1173
33.9599
65.3686
26.5434
60.085
66.2031
55.1082
53.5542
28.1705
Average
Percentage
Live Cells
87.267
STD
4.4391
32.4214
5.4841
53.6538
4.6907
TABLE 10 (continued)
Treatment Type
Microspheres without
Chemotherapeutics
Microspheres with
Chemotherapeutics
Microspheres with
Chemotherapeutics +
Magnetic Field
Total
Tumor
Area (µm2)
3458801
2449912.8
2593785
2978922
3098511
3493042
3470712
1984066
1063623
2947139
3509448
2978337
Area of Live
Cancer Cells
(µm2)
1514495.845
1425608.337
1663459.486
299189.163
454855.6392
1012975.519
532871.988
446055.7353
136077.9952
882012
701457.2444
869866.3882
99
Percentage
Live Cells
43.7867
58.1902
64.1324
10.0435
14.6798
28.9998
15.3534
22.4819
12.7938
29.9277
19.9877
29.2064
Average
Percentage
Live Cells
19.1829
24.5971
STD
3.2575
6.5187