DEVELOPMENT AND EVALUATION OF BIODEGRADABLE POLYMERIC NANOPARTICLES FOR CHEMO AND PEPTIDE
DELIVERY IN CANCER THERAPY
RAJ KUMAR SINHA
CENTRE FOR BIOMEDICAL ENGINEERING INDIAN INSTITUTE OF TECHNOLOGY DELHI
OCTOBER 2016
©Indian Institute of Technology Delhi (IITD), New Delhi, 2016
DEVELOPMENT AND EVALUATION OF BIODEGRADABLE POLYMERIC NANOPARTICLES FOR CHEMO AND PEPTIDE
DELIVERY IN CANCER THERAPY
by
Raj Kumar Sinha
Centre for Biomedical Engineering
Submitted
in fulfilment of the requirements of the DOCTOR OF PHILOSOPHY
to the
INDIAN INSTITUTE OF TECHNOLOGY DELHI
October, 2016
Dedicated to My
Family
CERTIFICATE
This is to certify that thesis entitled “Development and Evaluation of Biodegradable Polymeric Nanoparticles for Chemo and Peptide Delivery in Cancer Therapy” submitted by Mr. Raj Kumar Sinha to the Indian Institute of Technology Delhi for award of degree of Doctor of Philosophy, in Biomedical Engineering is a record of bonafide research work carried out by him. Mr. Raj Kumar Sinha has worked under our guidance and supervision and has fulfilled the requirement for the submission of the thesis.
The results contained in this thesis are original and have not been submitted in partial or full, to any other university or institute for the award of any degree or diploma.
(Dr. Harpal Singh) Professor Centre for Biomedical Engineering Indian Institute of Technology Delhi
Hauz Khas, New Delhi-110016
ACKNOWLEDGEMENTS
Pride, praise and perfection belong to Almighty alone, so first of all, I would like to offer my heartiest salutation at the lotus feet of the Supreme Being for the physical and mental strength bestowed upon me and in whose faith I was able to complete this task.
I wish to place on record my deep sense of gratitude and feeling of reverence to my thesis supervisor Prof. Harpal Singh, Centre for Biomedical Engineering, Indian Institute of Technology, Delhi for his guidance, constant inspiration, invaluable suggestions, broad vision and constructive criticism during the course of research work. It is more than mere formality that I express my heartfelt gratitude for his understanding and generosity bestowed on me without which this thesis would have been an uphill task.
I feel elated in acknowledging the precious help, suggestions and support by Dr. T.G. Shrivastava, NIHFW who helped me to accomplish this arduous work.
It’s my pleasant duty to convey my affectionate thanks to Dr. Manoj Gautam, Mr.
Dinesh Kumar, Mr. Manu Dalela, Mr. Vivek Bansal, Mr. Sumit Kapoor, Dr. Gurpal Singh, Dr. Lomas Tomar & Mrs. Dikshi Gupta, Sita Ram ji, Rajesh ji, Dr. Sruti Chatopadhyay, Dr. Swati Jain, Dr. Priyanka Tyagi, Mrs. Vasundhra, Mrs. Avneet, Ms.
Prabhjot (CBME,IIT Delhi), and all other friends and colleagues for their help and support during my research work. My heartfelt thanks are due to all the staff members of the Centre for Biomedical engineering, especially Mr. Rajesh, for their help during the research work.
I am thankful to INMAS, New Delhi and Directorate General of Health Services, Ministry of Health & Family welfare, New Delhi, for providing me necessary facilities to carry out the research work.
Finally, I am also thankful to my near and dear ones, without their affection, constant encouragement, blessings and support; I would not have reached this pinnacle in life.
Above all, I am grateful to the innocent mice who have given their lives for the success of this work.
(Raj Kumar Sinha
)
ABSTRACT
Cancer remains one of the world’s most fatal diseases; with about 12.7 million new cases and 7.6 million cancer death every year worldwide. Nanotechnology has tremendous potential to make an important contribution in cancer prevention, diagnosis and treatment. Paclitaxel has been shown to exhibit significant anticancer activity in various cancers like cell lung cancer, head and neck carcinomas and in various solid tumors, including ovarian, breast, etc. Use of paclitaxel is hindered due to its high toxicity, reduced bioavailability, non specificity and poor solubility in aqueous solution. In the present study, polylactic acid (PLA) based copolymers were studied as drug delivery system for cancer therapy. PLA72K-PEG4K block copolymers were successfully synthesized by conjugating PLA72K with PEG4K by Steglich esterification reaction using N, N, Dicyclohexylcarbodiimide (DCC) and 4-Dimethyl aminopyridine (DMAP). PLA72K-PEG4K-FA was also prepared by conjugating folic acid (FA) with PLA72K-PEG4K. Characterization of the synthesized block copolymers was done by FTIR and NMR spectroscopy. PLA showed Mw of 72487, on coupling with PEG (4 kDa) Mw increased to 78416 as measured by GPC confirming that single block of PEG4K was coupled with single block of PLA72K to give a PLA72K-PEG4K diblock.
Preparation of PTX loaded PLA72K-PEG4K and PLA72K-PEG4K-FA nanoparticles was performed using double emulsion solvent evaporation method and drug loaded polymeric nanoparticles (NPs) were characterized by DLS and SEM. The particle size of PLA, PLA72K-PEG4K and PLA72K-PEG4K-FA NPs was found to be 125± 3.6 nm, 114± 3.7nm and 110± 2.5 nm and increased upto 132 ±4.5 nm, 122± 4.6 nm and 124±
4.8 nm respectively after loading of PTX. Encapsulation efficiency of the PLA72K- PEG4K-FA nanoparticles is slightly less (83.93%) than that obtained with the PLA72K- PEG4K nanoparticles (87.53 %). In vitro release of PTX from PLA72K–PEG4K and
PLA72K-PEG4K-FA nanoparticles were 47% and 43% over 7 days, 79% and 72% over 60 days time period. Rate of release is slightly less in PLA72K-PEG4K-FA probably due to hydrophobic aromatic structure of folic acid present on the surface of NPs. In-vitro cellular uptake of PLA72K-PEG4K and PLA72K-PEG4K-FA nanoparticles in MCF-7 cells was performed with rhodamine B (RhB) loaded NPs for 6 hrs and 9 hrs, indicated more efficient cellular uptake by folate receptors mediated endocytosis in these cells. The cell viability of PTX-PLA72K-PEG4K and PTX-PLA72K-PEG4K-FA nanoparticles was evaluated in various cancer cell lines using MTT assay with PTX concentration (PTX) in the range of 0.01−10 μg/ml upto 72 hrs. The inhibitory concentration (IC50) for PTX- PLA72K-PEG4K and PTX-PLA72K-PEG4K-FA was very low for MCF-7 cells line approximately 100ng/mL and 10 ng/ml at 72hrs respectively, while in A549 inhibitory concentration (IC50) was around 1000 ng/ml at 72 hrs respectively.Confocal microscopy and flow cytometry results further supported the cellular uptake and apoptosis of cancer cells with PTX-PLA72K-PEG4K and PTX-PLA72K-PEG4K-FA nanoparticles. Administration of 60 mg/kg PTX equivalent of PTX-PLA72K-PEG4K and PTX-PLA72K-PEG4K-FA nanoparticles by intraperitoneal injection at 1st, 8th, 15th, and 23rd day in EAT tumor-bearing syngeneic BALB/c mice showed significant tumor growth inhibition with improved apoptosis effects in comparison with Taxol® without showing any cytotoxicity. On the basis of preliminary results, no subacute toxicity was observed in major organs, tissues and hematological system up to a dose of 60 mg/kg body weight in mice. PTX-PLA72K-PEG4K-FA nanoparticles showed 95% tumor inhibition without any relapse and may be considered as an alternative nanodrug delivery system for delivery of PTX in solid tumor.
MUC1 oncoprotein plays an important functional role in the development of human breast and other types of cancers. GO-203 is a 16 amino acid peptide (sequence from
MUC1-CD domain) with a potential anti-cancer activity. GO-203 therapeutic peptide is easily degraded by proteolytic degradation and showed short circulation half-life. These drawbacks can be overcome by using nanoparticles nanoplatforms and delivery system may also allow higher dosing of GO-203 with less pronounced anaphylactoid reactions often associated with intravenous delivery of protein therapeutics. Diblock PLA72K- PEG4K and tetrablock PLA72K-(PEG-PPG-PEG)12.5K were synthesized by Steglich esterification reaction and GO-203 loaded peptide NPs with 65% loading efficiency were prepared by double emulsion solvent evaporation technique. In terms of cumulative release, over 50% of the encapsulated GO-203 was released by day 7, prolonged and sustained release of GO-203 was observed till 60 days from both the NPs. However, the release profile of GO-203 from PLA72K-(PEG-PPG-PEG)12.5K nanoparticles was slower and more sustained than PLA72K-PEG4K at pH 7.4 due to hydrophobic nature of the PPG in (PEG-PPG-PEG)12.5K block. Rhodamine B loaded NPs showed significant uptake in MCF-7 cells from 3 to 12 hrs as confirmed by Confocal Laser Scanning Microscope (CLSM) studies indicated significant cellular uptake of NPs in MCF-7 cells. Confocal microscopy and flow cytometry results further supported the cell uptake and apoptosis of cancer cells with GO-203-PLA72K-(PEG- PPG-PEG)12.5K nanoparticles. Taken together, these findings indicate that encapsulation of GO-203 in polymeric NPs is an effective approach for the delivery of GO-203 to cancer cells. Administration of GO-203-PLA72K-(PEG-PPG-PEG)12.5K nanoparticles at a dose of 20 mg/kg weekly intraperitoneal injection for 3 weeks showed significant tumor growth inhibition in vivo in EAT and xenograft tumor model. These studies confirm the strong potential of PLA72K-(PEG-PPG-PEG)12.5K nanoparticles as a vehicle for delivery of anticancer peptides.
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CONTENTS
Page
No.
Chapter I: Introduction and literature review 1-46
1.1 Introduction 1
1.2 Cancer 3
1.2.1 Hallmarks of Cancer 4
1.3 Physiopathological characteristics of tumor 6
1.3.1 Leaky vasculature 6
1.3.2 Impaired lymphatic drainage 7
1.3.3 Acidic tumor microenvironment 7
1.3.4 Overexpression of growth factor receptors 7
1.4 Techniques used in cancer treatment 8
1.4.1 Surgery 8
1.4.2 Radiation therapy 8
1.4.3 Chemotherapy 9
1.4.4 Immunotherapy 10
1.5 Nanomedicine in cancer therapy 10
1.6 Mechanism of drug targeting 11
1.6.1 Passive drug targeting 11
1.6.2 Active drug targeting 12
1.7 Type of targeting moieties 14
1.7.1 Antibody based targeting for cancer 14
1.7.2 Peptide based targeting 14
1.7.3 Small molecule based targeting 15
1.7.4 Aptamer based targeting 15
1.7.5 Other targeting agents 15
1.8 Methods of conjugating targeting agents on to nanoparticles 16
1.8.1 Direct conjugation 17
ii
1.8.2 Conjugation with linker 17
1.9 Nanomaterials used for cancer therapy 18
1.9.1 Liposomes 18
1.9.2 Dendrimers 18
1.9.3 Metal based nanoparticles 19
1.9.4 Micelles based nanoparticles 19
1.9.5 Polymeric nanoparticles 20
1.9.5.1 Nanoparticles from natural polymers 21
1.9.5.2 Nanoparticles from synthetic polymers 22
1.10 Novel anticancer therapeutics 23
1.10.1 Peptides interfere with proliferative signals transduction cascades 23 1.10.2 Peptide inhibitors of cell cycle progression 24
1.10.3 Apoptosis Inducing Peptides 24
1.11 Characteristics of nanoparticles for cancer therapy 26 1.12 Nanoparticles under clinical investigation in cancer therapy 27
1.13 Rationale of the work 29
1.14 References 34
Chapter II : Preparation , characterization and biological evaluation of
biodegradable polymeric nanoparticles for delivery of chemo drug (paclitaxel)
47-104
2.1 Introduction 48
2.2 Experimental
2.2.1 Materials and Methods 52
2.2.1.1 Materials 52
2.2.1.2 Cell culture and maintenance 53
2.2.1.3 Animal model and treatment 53
2.2.2 Synthesis of PLA-PEG block copolymer and conjugation of folic acid (FA)
53
2.2.3 Characterization of PLA-PEGblock copolymer. 54 2.2.3.1 Fourier Transform Infrared Spectrometry (FTIR) 54
iii
2.2.3.2 Nuclear Magnetic Resonance spectroscopy (NMR) 54 2.2.3.3 Determination of molecular weight of PLA-PEGblock
copolymer by GPC
54
2.2.4 Preparation of Paclitaxel (PTX) and Rhodamine B loaded nanoparticles
55
2.2.5 Characterization of PTX-PLA-PEG nanoparticles 55 2.2.5.1 Dynamic light scattering (DLS) and zeta potential (δ)
measurement
55
2.2.5.2 Scanning electron microscopy 56
2.2.6 Encapsulation efficiency of PLA-PEG nanoparticles 56 2.2.7 In vitro release of PTX from PTX-PLA-PEG nanoparticles 56 2.2.8 Stability studies of PTX-PLA-PEG /PTX- PLA-PEG-FA
nanoparticles
57
2.2.9 In vitro studies of PTX-PLA-PEG nanoparticles 57 2.2.9.1 In vitro cellular uptakes studies of PLA-PEG NPs using
florescence imaging
57
2.2.9.2 In vitro cytoxicity studies assay of PTX-PLA-PEG NPs 58 2.2.9.3 Apoptosis assessment of PTX-PLA-PEG NPs 59
2.2.9.3.1 Apoptosis assay for assessment by AnnexinV–FITC
59
2.2.9.3.2 Mitochondrial membrane potential studies with PTX-PLA-PEG NPs
59
2.2.9.3.3 Reactive oxygen species (ROS) production studies with PTX-PLA-PEG NPs
60
2.2.9.3.4 Immunoblotting studies with PTX-PLA- PEG nanoparticles
61
2.2.10 In vivo Studies of PTX-PLA-PEG NPs 62
2.2.10.1. Hemolysis test 62
2.2.10.2 Toxicity assessment of PTX-PLA-PEG NPs in Balb-c mice
63
2.2.10.3 Ehrlich solid tumor regression studies of PTX-PLA- PEG NPs
63
2.2.10.4 Histology and immunohistochemistry of PTX-PLA- PEG NPs
65
iv
2.5.10.4.1 Tunnel assay 66
2.3 Results and Discussion 67
2.3.1 Synthesis of PLA-PEG block copolymer 67
2.3.2 Characterization of PLA-PEG block copolymer 67 2.3.2.1 Fourier transform infrared (ATR-FTIR) spectroscopy 67 2.3.2.2 Nuclear magnetic resonance (NMR) spectroscopy 68 2.3.2.3 Determination of molecular weight of PLA-PEG block
copolyme
70
2.3.3 Characterization of PTX-PLA-PEG nanoparticles 70 2.3.3.1 Particle size and zeta potential (δ) measurements 70 2.3.3.2 Scanning electron microscopy (SEM) 71
2.3.4 Encapsulation efficiency of PLA-PEG NPs 71
2.3.5 In vitro PTXfrom PTX-PLA-PEGnanoparticles 72
2.3.6 Stability studies of PTX- PLA-PEG NPs 73
2.3.7 In vitro Studies 74
2.3.7.1 Cellular uptake of PLA-PEG NPs using florescence imaging
74
2.3.7.2 Proliferation-Inhibition of folate positive MCF-7 and negative A549 cells by PTX-PLA-PEG NPs
76
2.3.7.3 Apoptosis studies with PTX-PLA-PEG NPs 79 2.3.7.3.1 Apoptosis studies by AnnexinV–FITC
with PTX-PLA-PEG NPs
79
2.3.7.3.2 Acridine Orange/Ethidium Bromide doubles staining (AO/EB) studies for apoptosis with PTX-PLA-PEG NPs
80
2.3.7.3.3 Mitochondrial membrane potential studies with PTX-PLA-PEG NPs
82
2.3.7.3.4 ROS production with PTX-PLA-PEG NPs
84
2.3.7.3.5 Induction of DNA fragmentation by PTX- PLA-PEG NPs
85
2.3.7.4 Evaluation of activation of apoptotic marker Bax by PTX-PLA- PEG NPs
87
v
2.3.8. In vivo Studies 87
2.3.8.1 Hemolysis study 87
2.3.8.2 In vivo Tumor inhibition studies with PTX-PLA-PEG NPs
89
2.3.8.3 Effects of PTX-PLA-PEG-FA NPs on hematological and biochemical parameters
93
2.3.8.4 Histology and immunohistochemistry of PTX-PLA- PEG NPs
94
2.3.8.5 Antitumor efficacy of PTX-PLAPEG 97
2.4 Discussion 97
References 100
Chapter III : Preparation , characterization and biological evaluation of biogedradable nanoparticles for delivery of anticancer peptide (GO-203)
105-149
3.1 Introduction 106
3.2 Experimental 110
3.2.1 Materials and methods 110
3.2.2 Synthesis of PLA-PEG and PLA-PEG-PPG-PEG block copolymers
112
3.2.3 Characterization of PLA-PEG and PLA-PEG-PPG-PEG block copolymers
112
3.2.3.1 Nuclear Magnetic Resonance spectroscopy (NMR) 112 3.2.3.2 GPC of PLA-PEG and PLA-PEG-PPG-PEG block
copolymers
112
3.2.4 Encapsulation of GO-203 in PLA-PEG and PLA-PEG-PPG-PEG NPs
113
3.2.5 Characterization of PLA-PEG and PLA-PEG-PPG-PEG NPs 113 3.2.5.1 Dynamic Light Scattering (DLS) and zeta potential
measurement
113
3.2.5.2 Encapsulation efficiency of PLA-PEG and PLA-PEG- PPG-PEG NPs
114
3.2.6 In vitro release GO-203 release studies from GO-203 NPs 114
3.2.7 In-vitro studies of PLA-PEG formulations 115
vi
3.2.7.1 Cell culture studies 115
3.2.7.2. In vitro cellular uptakes studies using florescence imaging
115
3.2.7.3. Acridine Orange/Ethidium Bromide double staining of GO-203 NPs
116
3.2.7.4. Mitochondrial membrane potential study of GO-203 NPs
116
3.2.7.5 ROS production study with GO-203 NPs 117 3.2.7.6 Cell viability assay of GO-203 NPs 117 3.2.7.7 Immunoblotting studies of GO-203 NPs 118 3.2.7.8 NADPH and GSH levels studies of GO-203 NPs 118 3.2.7.9 Colony formation assays with GO-203 NPs 118 3.2.7.10 Tumor sphere studies with GO-203 NPs 119 3.2.8 In-vivo tumor growth inhibition studies with GO-203 NPs 119
3.3 Results 120
3.3.1 Preparation of PLA-PEG and PLA-PEG-PPG-PEG block copolymer
120
3.3.2. Characterization of PLA-PEG and PLA-PEG-PPG-PEG block copolymers
120
3.3.2.1 NMR analysis for PLA-PEG and PLA-PEG-PPG-PEG block copolymers
120
3.3.2.2 Gel permeation chromatography (GPC) for PLA-PEG and PLA-PEG-PPG-PEG block copolymers
122
3.3.3 Characterization of PLA-PEG and PLA-PEG-PPG-PEG nanoparticles
122
3.3.3.1 Assessment of Dynamic Light Scattering (DLS) and Zeta Potential (δ) measurement
122
3.3.4 In vitro release of G0-203 from G0-203 NPs 123
3.3.5. In vitro studies of G0-203 NPs 125
3.3.5.1 In vitro cellular uptakes studies using florescence imaging
125
3.3.5.2 Acridine Orange/Ethidium Bromide doubles staining with GO-203 NPs
126
vii
3.3.5.3 Mitochondrial membrane potential analysis with GO- 203 NPs
127
3.3.5.4 ROS production with GO-203 NPs 128
3.3.5.5 DNA fragmentation analysis with GO-203 NPs 130 3.3.5.6 Survival studies of breast cancer cells with GO-203 NPs 131 3.3.5.7 Redox balance studies in breast cancer cells with GO-
203 NPs
133
3.3.5.8 Studies of effectiveness against NSCLC cells with GO- 203 NPs
135
3.3.5.9 Inhibition studies of breast and lung cancer cell with GO-203 NPs
136
3.3.6 In-vivo tumor growth inhibition studies with GO-203 NPs 138
3.4 Discussion 140
References 145
Chapter IV : Summary, conclusions and scope for future work 150-154
4.1 Summary and conclusion 150
4.2 Scope for the future work 154
viii
LIST OF FIGURES
Fig. No. Description Page No.
1.1 Normal and cancer cell division 5
1.2 The Hallmarks of Cancer 6
1.3 Schematic representation of the role of enhanced permeability and retention effect (EPR) in the delivery of drug carriers
13
2.1 FTIR Spectra of spectra of (i) PLA72K (ii) PLA72K-PEG4K-(iii) PLA72K-PEG4K- FA
68
2.2 Proton NMR spectra of (i) PLA72K (ii )PEG4K (iii) PLA72K- PEG4K (iv )FA (iii) PLA72K-PEG4K-FA,(vi)Enlarge spectra of showing peaks folic acid.
69
2.3 Scanning electron micrograph (SEM) of (A) PLA72K-PEG4K, (B) PLA72K-PEG4K -FA, (C) PTX- PLA72K-PEG4K and (D) PTX-PLA72K-PEG4K -FA
72
2.4 Cumulative release of PTX from PLA72K-PEG4K-and PLA72K- PEG4K-FA was studied in phosphate buffer at pH 7.4
73
2.5 Confocal Laser Scanning Microscope (CLSM) image of cellular uptake of Rhodamine B loaded PLA-PEG and PLA- PEG-FA nanoparticles in MCF-7 cell
75
2. 6 Prolihibition inhibition studies of PTX loaded nanoparticles on folate positive MCF-7cells
77
2.7 Prolihibition inhibition studies of PTX loaded nanoparticles on folate negative A549 cells
78
2.8 Apoptosis assay to detect membrane integrity by AnnexinV–
FITC on treatment with different PTX loaded nanoparticle after incubating in folate positive (MCF-7) and folate negative (A549) cells using flow cytometry ,
80
2.9 Confocal laser scanning microscopic images of Acridine Orange/Ethidium Bromide doubles staining of MCF-7 and A549 cells treated with PTX, PTX-PLA72K-PEG4K,and PTX- PLA72K-PEG4K -FA
81
2.10 Confocal laser scanning microscopic images of JC-1 dye staining of in MCF-7 cells treated with PTX, PTX-PLA72K- PEG4K,and PTX-PLA72K-PEG4K-FA
83
2.11 Flow cytometry data of MCF-7 cells stained with JC-1 dye 84
ix
Fig. No. Description Page No.
2.12 Flow cytometry data of MCF-7 cells stained with 10-Nonyl acridine orange (NAO)
85
2.13 Flow cytometry data of MCF-7 cells stained with Hydroethidine (HE)
86
2.14 Immuno-detection of apoptotic protein Bax in MCF-7 cells on treatment PTX, PTX-PLA72K-PEG4K and PTX-PLA72K- PEG4K–FA for 72 hr
87
2.15 Effects of PTX loaded NPs on red blood cell aggregation was monitored by (i) bright field microscopy (ii) UV-visible spectroscopy
88
2.16 In vivo tumor efficacy after treatment of PTX-PLA72K-PEG4K, PTX-PLA72K-PEG4K–FA NPs and Paclitaxel (Taxol®) on EAT bearing syngeneic BALB/c mice
91
2.17 In vivo body weight changes (%) after treatment of PTX- PLA72K-PEG4K, PTX-PLA72K-PEG4K–FA nanoparticles and Taxol® on EAT bearing syngeneic BALB/c mice
92
2.18 High power photomicrographs of H&E staining of excised tumor tissues treated with Taxol® and PLA-PEG-PTX, and PLA-PEG-FA-PTX nanoparticles for 40 days. (Black arrow shows single apoptotic body).
96
2.19 High power photomicrographs of H&E and immunohistochemical staining of treated with PTX-PLA72K- PEG4K and PTX-PLA72K-PEG4K-FANPs for 35 days.
96
2.20 Kaplan-Meier survival curve of EAT tumor bearing syngeneic BALB/c mice treated with different PTX formulations (n=6)
97
3.1 C terminal MUC1 peptide, target peptide GO-203 and control peptide CP-2 sequences
109
3.2 Proton NMR spectra of (i) PLA (ii) PEG –PPG-PEG (iii) PLA-PEG-PPG-PEG
121
3.3 Cumulative release of G0-203 from PLA-PEG and PLA-PEG- PPG-PEG nanoparticles was studied in phosphate buffer at pH 7.4
124
3.4 Confocal Laser Scanning Microscope (CLSM) image of cell uptake of Rhodamine B loaded PLA72K-(PEG-PPG-
PEG)12.5Knanoparticles in MCF-7 cell. (Scale bar 20 m)
125
x
Fig. No. Description Page No.
3.5 Confocal laser scanning microscopic images of Acridine orange/Ethidium bromide double staining in MCF-7 cells treated with GO-203 NPs
127
3.6 Confocal laser scanning microscopy of JC-1 dye staining MCF-7 cells treated with GO-203 NPs.
128
3.7 Assessment of reactive oxygen species (ROS) production by 10-NAO after treatment GO-203 NPs
129
3.8 Assessment of ROS production and DNA fragmentation by hydroethidine (HE) after treatment GO-203 NPs
130
3.9 Effects of GO-203/nanoparticles (NP) on viability of breast cancer cells.
132
3.10 Effects of GO-203/NPs on MUC1-C homodimerization and function in breast cancer cells.
134
3.11 Targeting MUC1-C with GO-203/NPs is effective against NSCLC cells.
136
3.12 Effects of GO-203/NPs on breast cancer cell self renewal 138 3.13 Tumor regression studies in EAT and xenograft tumor mice
treated with GO-203/NPs
140
xi
LIST OF TABLES
Table No. Description Page No.
1.1 Cell cycle inhibitory and pro-apoptotic peptides 25 1.2 Nanoparticle delivery systems on the market and in clinical
development
28
2.1 Protocol for treatment of different groups of EAT mice with nanoformualaton along with control
64
2.2 Molecular weights of the PLA72K and PLA72K-PEG4K by GPC
70
2.3 Particle size and zeta potential measurement of NPs 71
2.4 In vitro stability studies of PTX- PLA72K-PEG4K and PTX- PLA72K-PEG4KFA nanoparticles.
74
2.5 Blood cell counts after intraperitoneal treatment in EAT tumor bearing BALB/c mice.
93
2.6 Serum chemistry after intraperitoneal treatment in EAT tumor bearing BALB/c mice
94
3.1 Molecular weights of the PLA72K, PLA72K-PEG4K and PLA72K-(PEG-PPG-PEG)12.5K by GPC
122
3.2 Particle size and zeta potential measurement of NPs 123
xii
ABBREVIATIONS
% Percent
µg Microgram
µl Microliter
µm Micrometer
0C Degree centigrade
1H Proton
ALB Albumin
ALP Alkaline phopshatase ALT Alkaline aminotransferase
AQC AQC motif
AST Aspartate aminotransferase ATR Attenuated total reflectance BCA Bicinchonic Acid
BSA Bovine serum albumin BUN Blood Urea Nitrogen CD Clusture of differentiation CK Creatinine Kinase
cm-1 Per centimeter Conc. Concentration CP-2 Control Peptide – 2
CQC CQC motif
CR Controlled release CRE Creatinine
Da Dalton
DAMP 4-dimethylaminopyridine DCC dicyclohexylcarbodiimide DLS Dynamic light scattering
Dr. Doctor
EDC 1-Ethyl-3-[3-Dimethylaminopropyl] Carbodiimide Hydrochloride EDTA Ethylenediamine tetrapthalic acid
xiii
ELISA Enzyme linked immunosorbant assay EMEM Eagle’s minimal essential medium EP European Pharmacopoeia
EPI Expanded programme of immunisation FA Folic acid
FACS Fluorescence-activated cell sorting (FACS) FDA Food and drugs administration
Fig. Figure
FITC Fluorescein isocyanate FTIR Fourier transform infrared
g Gram
GLB Globulin
gms Grams
GP Group
GPC Gel permeation chromatography HBI Human Biological Institute HCl Hydrochloric acid
HPLC High performance liquid chromatography HRP Horse raddish peroxidase
hrs Hours
IL Interleukin
IU International Unit kDa Kilo Dalton
KV Kilo Volt
LALLS Left angle light scattering detector LD50 Lethal dose fifty
Mg Milligram
MHz Mega Hertz
ml Milli liter mmol Milli mol
Mn Number average molecular weight Mol. Wt. Molecular weight
xiv MTD Maximum tolerated dose MUC1-C Mucin 1 C-Terminal Subunit MUC1-CD Mucin 1 Cytoplasmic Domain MUC1-N Mucin 1 N-Terminal Subunit
mV Milli Volt
Mw Weight average molecular weight
mΩ Milli ohm
NA Not applicable NaOH Sodium hydroxide
ND Not done
NHS N-Hydroxysulfo Succinimide
nm Nanometer
NMR Nuclear magnetic resonance NPs Nanoparticles
NS Normal saline O/W Oil in water
PBS Phopsphate buffer saline PCL Polycaprolactone
PDI Polydispersity index PEG Poly ethelene glycol
pg Picogram
PGA Poly (glycolic acid) pH Potential of hydrogen PLA Polylactic acid
PLGA Poly D-Lactide-co-glycolide PTX Paclitaxel
Pvt. Private
RALLS Right angle light scattering detector rpm Revolutions per minute
RT Room temperature s/c Subcutaneous
SDS Sodium dodecyl sulphate
xv
sec Seconds
SEM Scanning electron microscope TBL Total bilirubin Level
T-cell Thymus derived cell
TEM Transmission electon microscope THF Tetrahydrofuran
TM Trade mark
TMB 3,3,5,5 Tetra methyl amino benizidine TP Total Protein
TUNNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling UIP Universal immunisation programme
US United States
USA United states of America UV Ultra violet
v/v Volume by volume w/v Weight by volume
WHO World Health Organisation WNT Wnt signal pathway
α Alpha
β Beta
γ Gamma
