Development of nanoplatforms for cancer diagnosis and therapy

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DEVELOPMENT OF NANOPLATFORMS FOR CANCER DIAGNOSIS AND THERAPY

By

MANOJ KUMAR

Centre for Biomedical Engineering

Submitted

infuiflulment ofthe requirements ofthe degree of

Doctor of Philosop 如

to the

INDIAN INSTITUTE OF TECHNOLOGY DELHI

October, 2012

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CERTIFICATE

This is to certify that the thesis entitled "Development of Nanoplatforms for Cancer Diagnosis and Therapy" submitted by Mr. Manoj Kumar to the Indian Institute of Technology, Delhi for the award of degree of Doctor of Philosophy, in Biomedical Engineering is a record of bonaffide research work ca市ed out by him. Mr. Manoj Kumar has worked under our guidance and supervision and has fuiffihled the requirement for the submission of this thesis.

OEe 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. Harpai Singh) Professor

(Dr. A.K.Dinda) Professor

Centre for Biomedical Engineering Department of Pathology

Indian Institute ofTechnology Delhi All India Institute of Medical Sciences, New Delhi Hauz Khas, New Delhi-110016 Ansari Nagar, New Delhi- i 10029

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ACKNOWLEb&EMENTS

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Abstract

Cancer remains one of the world's most deadly diseases; with about 12 million new cases and 7.6 million cancer death every year worldwide. Despite rapid advances in diagnostic procedures and treatments, the overall survival rate fflom cancer has not improved substantially over the past 30 years. Therefore, there is a need to develop novel approaches for the accurate detection of cancer at ari early-stage.

Nanotechnology has tremendous potential to make an important contribution in cancer prevention, diagnosis, imaging and treatment. Nanoparticles delivery systems can deliver a higher amount of anticancer drugs/imaging agents to the vicinity of the tumor, thus improving therapeutic effficacy and reducing harmful non-speciffic side effects. Thus nanotechnological approaches might be most effective and effficient means to confflont early diagnosis and treatment of cancer.

Tumor targetable, super paramagnetic iron oxide nanoparticles (SPIONs) capped with citric acid! 2-bromo-2-methyl propanoic acid (CA/BMPA) were developed which are compact, water dispersible, biocompatible with narrow range of size dispersity (7-10 nm) and relatively high T2 relaxivity (R2=222L.mmoF1 .sec-5. The targeting effficacy of unconjugated and folic acid (FA)-conjugated SPIONs was evaluated in folate receptor (FAR) overexpressing (MCF-7&HepG2) and negative tumor cell line (A549). FAR-positive cells incubated with FA-SPIONs showed much higher intracellular iron content without any cytotoxicity. In-vitro MRI studies on tumor cells showed better T2-weighted images in FA-SPIONs. These ffindings indicate that FA- CA/BMPA-SPIONs possess high colloidal stability with excellent sensitivity of imaging and can be a useful MRI contrast agent for the detection of cancer. FA- SPIONs were also evaluated for in-vivo pharmacokinetics, biodistribution and toxicity in mice. Bio-distribution studies revealed that presence of SPIONs in various

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body tissues changed with time but greater fflaction ofthe injected iron nanoparticles were observed to be localized in the liver and spleen compared to the brain, heart, kidney and lung. Changes in serum iron levels were analyzed upto 3 weeks aifier intravenous administration of SPIONs in mice and results showed a transient increase in serum iron level compare to control animal up to i week and then declinedaifierwards. FA-SPIONs did not show any toxicity at injected dose of 20mg/kg body weight in mice.

Superparamagnetic iron oxide nanoparticles were also evaluated as a delivery vehicle for anticancer peptide. CAIBMPA capped SPIONs were conjugated with L-form of anticancer peptide (NuBCP-.9) without cell penetrating peptide (CPP) which kills the cancer cells overexpressing Bdl-2. The NuBCP-9-SPIONs showed effective cell killing of cancerous cells and therapeutic effficacy of this nanoformulation is 2.5-5 time higher than that of CPP conjugated NuBCP-9. Complete inhibition in proliferation was observed in cancer cells due to activation cytochrome C3 dependent pathway of apoptosis. It was established that developed SPIONs can deliver L- form of NuBCP-9 without cell penetrating peptides and cause apoptosis in Bdl-2 overexpessing cell without losing its therapeutic efficacy. In-vivo studies of tumor regression in EAT mouse model showed 50% tumor regression in animals treated with NuBCP-9-SPIONs suggesting that these nanoparticles can successfully deliver the labile L-form of the NuBCP-9 to the tumor tissue without loss in its activity.

Prussian blue staining of the tumor tissue also conffirmed the presence of SPIONs. To conclude, d四elone'i n麺(na- J.- -亜cies nos---- 肥ss hoth fhe nr-- - - -- ---I--_叩erH s ofiniwnt n-i ApIF'ipr',-- - --- --- ---- . --- of anticancer peptide and hence can be used as theragnostic (multimodal) agents for early cancer detection and treatment with huge clinical translation capabilities.

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Polylactic acid (PLA) and polyethylene glycol (PEG) based diblock copolymers were synthesized and characterized by various techniques viz. NMR and GPC.

Nanoparticles were prepared from these block copolymers and investigated as a carrier for delivery of L- form of anticancer peptide 倒uBCP-9) without cell penetrating peptide (CPP). NuBCP-9 loading was simultaneously achieved with particle synthesis by double emulsion precipitation technique. The effect of varied PEG chain lengths on particle size and on loading effficiency of NuBCP-9 was determined using BCA assay kit. PLA-PEG4000 showed highest loading effficiency of 65.1%while lowest encapsulation effficiency of 52.09% was observed in PLA-PEG i 000 NPs. In-vitro NuBCP-9 release studies performed under simulated conditions (with different buffers and pH) showed initial burst release followed by near ffirst order release kinetics. Maximum peptide release of about一90% was observed in PBS buffer at pH 7.4 while it was 68% only in pH 5.0 over a period of 60 days. In-vitro proliferation inhibition studies were performed in multiple cancer cell lines along with the normal cells. NuBCP-9 loaded PLA-PEG nanoparticles showed 100%

proliferation i山ibition in cancer cell with a minimum 24 h delay while no inhibition was observed in normal cells. Therapeutic potential of NuBCP-9 loaded nanoparticles was found to be 2-3 folds higher than that ofNuBCP-9 conjugated with CPP. In-vivo toxicity studies of the peptide loaded nanoparticles showed no toxicity at a dose of i 00mg/kg body weight of the nanoformulations with 10%peptide concentration.

Erlich Ascetic Tumor regression studies revealed the complete regression of tumor in 21 days in Balb-c mice treated with NuBCP-9 loaded PLA-PEG nanoparticles without reoccurrence at a dose of 20mg/kg body weight of NuBCP-9 peptide.

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Chapter II: Synthesis. characterization and biolo2ical evaluation of iron oxide nanoparticles

2.1 Introduction 67

2.2

2.2.1 2.2.2

2.2.3

2.2.4

2.2.5

Experimental

Materials and Methods

Synthesis of superparamagnetic iron oxide nanoparticles

(SPIONs)

Ligand exchange ofoleic coated SPIONs with tetramethyl ammonium hydroxide (TMAOH)

Ligand exchange ofoleic coated SPIONs with dimercaptosuccinic acid (DMSA)

Preparation of citric acid/2-bromo-2-methylpropionic acid 7.6 Nanomaterials cancer therapy

References

PART-A: Synthesis, Characterレation and in-vitro evaluation of iron oxide nanoparticles

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(CA/BMPA) coated SPIONs

Bioconjugation of SPIONs nanoparticles with folic acid (FA) Characterization of Iron oxide nanoparticles

Transmission electron microscopy (TEM)

X-ray diffflaction (XRD) pattern of iron oxide nanoparticles Superconducting quantum interference device (SQUID) magnetometry

Attenuated total reflection-Fourier transform infflared spectroscopy (ATR-FTIR)

In-vitro studies

Cellular Interaction studies of iron oxide nanoparticles Prussian blue staining

Transmission electron microscopic study XTT Assay

Cellular uptake study Statistical analysis Results and discussion

Synthesis of hydrophobic SPIONs

Characterization of hydrophobic SPIONs Transmission electron microscopy (TEM)

2.3.2.2 X-ray diffflaction (XRD) pattern ofSPIONs and single area ei ectron di冊action pattern

Superconducting quantum interference device (S QUID) magnetometry

Attenuated total reflection-Fourier transform infflared 2.2.6

2.2.7 2.2.7.1 2.2.7.2 2.2.7.3

2.2.8 2.2.8.1 2.2.8.2 2.2.8.3 2.2.8.4 2.2.8.5 2.2.8.6 2.3

2.3.1 2.3.2 2.3.2.1 2.3.2.2

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spectroscopy (ATR-FTIR)

2.3.3 Generation of SPIONs suitable for biomedical applications by ligand exchange

2 .3 .4 Characterizations

2.3.4.1 Transmission electron microscopy (TEM)

2 .3 .4. 2 Attenuated total reflection-Fourier transform infflared spectroscopy (ATR-FTIR)

2.3.5 Bioconjugation ofCAIBMPA-SPION with folic acid (FA) 2.3.6 Cellular interaction studies of SPIONs

2.3.6.1 Prussian blue staining

2.3.6.2 Transmission Electron Microscopic (TEM) Studies 2.3.6.3 XTT Assays

2.3.6.4 Concentration dependent cellular uptake studies 2.3.6.5 Time dependent cellular uptake studies

PART B: In-vivo evaluation of FA-SPIONs 2.4 Material and Methods

2.4.1 Materials 2.4.2 Animal Studies

2.4.3 Sample collection for biodistribution studies 2.4.4 Measurement of serum iron level

2.4.5 Analysis oftissue sample for Fe concentration 2.4.6 Histopathological studies

2.4.7 Transmission electron microscopy (TEM) studies 2.4.8 Toxicity study in mice

2.4.8.1 Analysis ofblood and serum

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2.4.9 Statistical analysis 2.5 Results and discussion

2.5.1 Measurement ofserum iron levels

2.5.2 Biodistribution ofFA-SPIONs by analyzing Fe concentration in different organs

2.5.3 Histopathological studies

2.5.4 TEM studies oftissue samples for FA-SPIONs 2.5.5 In-vivo toxicity assessment ofFA-SPIONs in mice 2.5.5. 1 Animal behavior study

2.5.5.2 Hematological studies 2.5.5.3 Biochemistry panel assay 2. 5 . 5 .4 Histopathological studies

2.6 Conclusion

References

Chapter III: Evaluation oflron Oxide Nanoparticles as MlU Contrast Agents and Delivery Vehicle for Anticancer Peptide 3 . 1 Introduction

PART A: Evaluation of Iron Oxide Nanoparticles as Magnetic Resonance Imaging Contrast Agents

3.2 Experimental

3.2.1 Materials and Methods ,,, r':1。i Materials

3.2.1.2 Cell Culture.

3.2.2 In-Vitro MR Imaging.

3.2.2.1 Preparation ofphantom agar gels for imaging

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3.2.2.2 Preparation ofMCF-7 for MRI studies I 45 3 .2.2.3 Measurements of imaging characteristics of magnetic

nanoparticles in phantom gels and MCF-7 cells. I 46

3 .3 Results and Discussion I 47

3.3.1 MRI characteristics ofmagnetic nanoparticles in phantom agar

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PART B: Evaluation of Iron Oxide Nanoparticles as delivery vehicle for

anticancer peptide 151-174

3 .4 Experimental 151

3.4.1 Materials and Methods 151

3.4.1.i Materials 151

3.4.1.2 Animal Studies 151

3.4.2 Methods 151

3.4.2.1 Bioconjugation ofSPIONs with anticancer peptide (NuBCP-9) 151

3.4.2.2 Characterization of NuBCP-9-SPIONs 152

3.4.2.2.1 Transmission electron microscopy (TEM) 152 3 .4.2.2.2 Attenuated total reflection-Fourier transform infflared

spectroscopy (ATR-FTIR) 152

3.4.2.3 Anticancer peptide immobilization efficiency of SPIONs 153 3.4.2.4 In-vitro cell culture studies of NuBCP-9-SPIONs. 153 3.4.2.4.1 Proliferation inhibition studies ofNuBCP-9 in difたrent cell lines 154 3.4.2.4.2 Apoptosis assay by Annexin V-alexa fluor 488/PI staining 154 3.4.2.4.3 Western blotting experiments ofcancer cells treated with

NuBCP-9-SPIONs 156

3.4.2.5 In-vivo studies 157

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3.4.2.5.1 Ehrlich solid tumor regression studies

3.4.2.5.2 Effect ofNuBCP-9-SPIONs on biochemical parameters 3.4.2.5.3 Histopathological studies

3.5 Results and discussion

3.5.1 Immobilization effficiency ofpeptide on SPIONs 3.5.2 Characterization of NuBCP-9-SPIONs

3.5.2.1 Transmission Electron Microscopy

3.5.2.2 ATR-FTIR spectroscopy ofpeptide immobilized SPIONs 3.5.3 Proliferation Inhibition studies ofcancer cell lines treated with

NuBCP-9-SPIONs

3.5.4 Apoptosis assay by Annexin V-alexa fluor 488/PI staining 3.5.5 Western blotting

3.5.6 Erlich Ascetic Solid Tumor regression study

3.5.7 Effect ofNuBCP-9-SPIONs on biochemistry panel assays 3.5.8 Histology studies oftumor tissue

3.6 Conclusion

References

Chapter IV: Polylactic acid-polyethylene glycol nanoparticles for delivery of anticancer peptide

4. 1 Introduction 4'2 Experimental

4.2.1 Materials and methods 4.2.1.i Materials

4.2.1.2 Animal studies

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4.2.2 Synthesis ofPLA-PEG block copolymer

4.2.2.1 Characterization of s押thesized PLA-PEG block copolymers 4.2.2.1 .i Nuclear Magnetic Resonance spectroscopy

4.2.2.1.2 Gel permeation chromatography ofPLA-PEG block copolymers 4.2.3 Encapsulation ofNuBCP-9 in PLA-PEG nanoparticles

4.2.3 . 1 Characterization of PLA-PEG nanoparticles

4.2.3.2 Dynamic light scattering (DLS) and zeta potential（ち）

measurement

4.2.3.3 Transmission electron microscopy (TEM)

4.2.4 Encapsulation effficiency ofNuBCP-9 loaded nanoparticles 4.2.5 In vitro NuBCP-9 release s加dies fflom PLA-PEG NP's 4.2.6 In-vitro studies ofPLA-PEG nanoformulations

4.2.6.1 Cell culture studies

4.2.6.2 Cytotoxicity assay ofPLA-PEG nanoparticles

4.2.6.3 In vitro cell uptake studies using fluorescence imaging

4.2 .6.4 Proliferation inhibition studies of NuBCP-9 encapsulated NP 's 4.2.6.5 Apoptosis assay by Annexin V-alexa fluor488/PI staining

4.2.6.6 Western blot studies of cancer cells treated with NuBCP-9-PLA- PEG NP's.

4.2.7 In-vivo studies

4.2.7.1 Toxicity assessment ofPLA-PEG NP's in Balb-c mice 4.2.7.2 Hematological studies

4.2.7.3 Biochemistry panel assay

4.2.7.4 Histopathological studies ofvital organs 4.2.7.5 Ehrlich solid tumor regression studies

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Effect ofNuBCP-9-PLA-PEG NP's on biochemical parameters Histopathological studies

Results and discussion

Synthesis ofPLA-PEG block copolいtiers

Characterization of PLA and PLA-PEG block copolymers NMR spectroscopy

Gel permeation chromatography (GPC) of PLA, PLA-PEG block copolymers

Characterization ofPLA and PLA-PEG nanoparticles Dynamic Light Scattering (DLS) and Zeta Potential（ち）

Measurement

Transmission Electron Microscopy of PLA-PEG nanoparticles Encapsulation effficiency of NuBCP-9 encapsulated

nanoparticles

In-vitro peptide release ifiom PLA-PEG nanoparticles In-vitro studies

Cytotoxicity of PLA and PLA-PEG nanoparticles In-vitro cell uptake studies using fluorescent imaging

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4.3.7.4 Histopathological studies 224

4.3.8 Tumor inhibition studies 226

4.3.8 Erlich ascetic solid tumor regression studies 226 4.3.8.2 Effect ofNuBCP-9-PLA-PEG NP's on biochemistry Panel

Assays 231

4.3.8.3 Histology studies oftumor tissue 232

4.4 Conclusion 234

References

Chapter V: Summary and scope for future work

Summary and conclusion

Scope for future work

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Development of nanoplatforms for cancer diagnosis and therapy