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IN VITRO EVALUATION OF NIGELLA SATIVA-BASED COLLOIDAL DRUG CARRIERS

FOR DELIVERY ACROSS BLOOD-BRAIN BARRIER

BY

NURUL HAFIZAH BINTI MOHD NOR

A thesis submitted in fulfilment of the requirement for the degree of Master of Pharmaceutical Science

(Pharmaceutical Technology)

Kulliyyah of Pharmacy

International Islamic University Malaysia

MAY 2015

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ii

ABSTRACT

Nigella sativa (N. sativa) oil is known to have neuroregenerative effect while contributing to lipophilicity of either the formulation or of the therapeutic carrier system. The aim of this study was to evaluate the in vitro effect of Nigella sativa- based colloidal drug carriers for delivery across the blood-brain barrier. Firstly, the poly (D, L-lactic-co glycolic acid) (PLGA)/chitosan/N. sativa nanoparticles were fabricated via optimized diffusion-solvent-evaporation method and were characterized in terms of their particle size, charge analysis and surface morphology. From the characterization tests, five fabricated nanoparticles were selected according to the particle size in the range of 10 to 970 nm with smooth external surface and stable surface charge for further investigation. Secondly, the N. sativa oil-in-water microemulsions were prepared via the drop-wise titration of pre-determined volumes of N. sativa oil into mixtures of surfactant blends (Span 20, Span 80, Tween 20, Tween 80, Tween 85) and water. All transparent ternary mixtures were characterized for their viscosity, droplet size, thermodynamically stable characteristics, optically transparent appearance and high solubilization capability. The stability of the microemulsion was evaluated by subjecting them to stressful conditions, namely centrifugation (2000 g for 20 minutes) and heating in a dry oven (60 to 105 °C for 5 hours) and the droplet size was determined following one month storage at room temperature (25°C ± 2) thereafter. Based on the results, a phase ternary diagram was constructed from corresponding volumes of those 3 components. N. sativa mixtures (ranging from 7.4 to 10.7%) prepared at HLB 16 of surfactant blends (Tween 20:Tween 80; 6:4) with deionized water (ranging from 17.9 to 18.5%) yielded transparent liquids. The constructed phase diagram displayed regions of a few types of microemulsions and emulsions. The droplet size of freshly prepared mixtures was wider in range (5 to 15.6 nm) than the size following stressful condition (11.3 to 12.4 nm). The data indicated that N. sativa oil could be formulated into oil-in-water microemulsions at specific HLB value of surfactant blends. Following characterization of these two carrier systems, four in vitro testing were performed: (1) cell viability on MDCK 1 and N2a cells, (2) neurite outgrowth induction on N2a cells (3) transfection efficiency on MDCK 1 and N2a cells and (4) permeability assay across blood-brain barrier following loading of the nanoparticles. The results showed that: (1) no significant cell toxicity was found when MDCK 1 cells were treated with nanoparticles even at 1000 µg/ml. The microemulsions were concluded to be very potent to N2a cells and worked in a dose-dependent manner; (2) small particle size and high encapsulation efficiency of N. sativa oil could be related to the effect of the neuroregenerative property of N. sativa oil on N2a cells, leading to neurite outgrowth;

(3) transfection efficiency assay indicated that the medium molecular weight of chitosan-bearing nanoparticle gave the highest transfection into the cells and (4) permeability assay showed the low molecular weight of chitosan-bearing nanoparticle gave the lowest transepithelial electrical resistance (TEER) value, indicated the highest permeation ability compared to the other nanoparticles. These systems were envisaged to enable rapid viewing of neurite extension on neuronal cell lines loaded with N. sativa oil. It was also suggested that N. sativa-based colloidal drug carrier would assist in future blood-brain barrier studies using non-viral gene therapy delivery system for neurodegenerative disease treatment.

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iii

ثحبلا ةصلاخ

في وتهماسبم ةيبصعلا ايلالخا جاتنا ىلع وتردقب فورعم ءادوسلا ةبلحا تيز نا ناوىدلل باذمذلاا ةيصاخ ةدايز

ةبحلل ةيورغلا ءاودلا تلاقان ايبرتمخ في مييقتلل ثحبلا اذى فدهي.بيكتًلل ةيئاودلا لماولحا وا بيكتًلل ءاوس ينستح برع ةيونانلا ةكبرلا ةبح و نازوتيشلا عم يج لب ليوب جاتنا تم لاوا .غامدلا في مدلا زجاح برع ءادوسلا تابيذلدا راشتنا ةقيرط في بي يوونلا ضملحا عضو تم ةقيرطلا هذى في .ةفلتمخ قرطو تانوكلدا مادختساب رخبتلا

لكشلا و تانحشلا ليلحتلا و مجلحا قيرط نع ةينونانلا تاميسلجا رايتخا تم دقو . ةينونانلا تاميسلجا لخاد في تاميسلجا مجلح اقفو ةيونان تابكرم ةسخم رابتخا تم تارابتخلاا هذى نم ,يحطسلا دودح

10 لىإ تًمونان

970 باث ةنحش و يجراخ حطس عم تًمونان ة

تيز دادعا تم ايناث . –

قيرط نع ةكبرلا ةبلح نشليما وركيام ءام

نابس( تنتكفرس في ةكبرلا ةبح تيز عضو 20

نابس ، 80 نيوت ، 20 نيوت ، 80 نيوت ، 85 لك .ءالداو )

رابتخا قيرط نع مىزيتد تم ةفافشلا ةيثلاثلا طيلاخلدا رهظم ،ةيارلحا ةاكيمانيدلا ،تايرطقلا مجحو ،ةجوزللا

درطلا يىو ،ةيساق فورظل مهضيرعت لبق نم نشليماوركيالدا رارقتسا مييقت تم .نابوذلا ةردقو ايرصب فافش ( يزكرلدا 2000

ةدلد مارج 20

جتلا نرف في ينخستلاو )ةقيقد ( فيف

60 لىإ 105 ةدلد ةيوئم ةجرد 5

جح ديدتح تم دقو )تاعاس ( ةفرغلا ةرارح ةجرد في نيزختلا نم دحاو رهش دعب تايرطقلا م

25 ةيوئم ةجرد ±

2 ( ةكبرلا ةبح بكرم .) 7.4

لىإ 10.7 يا لا بي يى في دعأ يذلا )٪

16 نيوت( عم 20

نيوت : 80 ؛ 6 :

4 ( تانويلأا عوزنم ءالدا عم ) 17.9

لىإ 18.5 نم تايرطقلا مجح ناكو .فافش لئاس جاتنا نع رفسأ )٪

اخلدا ( قاطنلا في عسوأ ةجزاطلا طيل 5

تيح 15،6 ( ةدهلمجا ةيلاتلا وتلاح مجح نم )تًمونان 11،3

- 12،4

تديش نيايبلا مسرلا ةلحرم .ةثلاثلا تانوكلدا كلت نم يطيطتخ مسر ءانب تم ،جئاتنلا هذى ىلع ءانبو .)تًمونان براتج ةعبرا ءارجا تم كلذ دعبو .نشليماوركيالداو نشليملاا نم عاوناب يىو ةيبرتمخ

1 يس يد ما ناتيللخا ةيلاعف )

نا و يك 2

فلا 2 نا ايلاخ ىلع فيرصلا روهظلل رشابلدا صحفلا قيرط ن ) 2

فلا 3 ناتيلخلل نشكفسنارت )

4 ( نأ نا جئاتنلا ترهظا .ةيونانلا تاميسلجا ليمتح دعب غامدلا في مدلا زجاح برع ةيذافنلا ) 1

روثعلا متي لم )

يربك ةيلخلل ةيسم يأ ىلع زيكرت ىلعأ دنع تىح ةيونانلا عم ايلالخا جلاع تم امدنع ة

1000 .لم / مارغوركيم

( 2 نا ةيللخ تياروينلا ءانب في دعاس ءادوسلا ةبلحا تيز نم ةيبصعلا ايلالخا جاتنا ةيصاخ نا ) 2

( .فلا 3 )

لخا لاك في نشيأكفسنرت ىلعأ ىطعأ نازوتيشلا نم طسوتلدا يئيزلجا نزولا نأ جئاتنلا تراشأ ( .ينتيل

4 رهظأ )

امد ،ليليثيباسنارت في لقا ةيئابرهكلا ةمواقلدا ةميق ىطعأ نازوتيشللال يئيزلجا نزولا ضافنخا نأ ةيذافنلا صحف طوطخ ىلع ايبرتمخ ةمظنلأا هذى رابتخا تم ،مومعلا في .غامدلا في مدلا زجاح برع ذافنلا ةردق عافترأ ىلع لدي ةبلحا تيزب ةلممح ةيبصعلا ايلالخا ةبلح ةيورغلا ءاودلا لقاون نأ اذبهو .نوىدلل ةبمح ىرخأ داوم ابمر وأ ءادوسلا

يرغ نييلجا جلاعلا ماظن مادختساب لبقتسلدا في غامدلا في مدلا زجاح ةسارد في دعاسي نأ ونأش نم ةكبرلا .ةيبصعلا ضارملأا جلاعلل يسويرفل

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iv

ABSTRAK

Minyak Jintan Hitam diketahui mempunyai kesan neuroregenerasi sambil memberikan lipofilisiti kepada formulasi mahupun kepada sistem pembawa ubat terapeutik. Kajian ini dijalankan bagi tujuan evaluasi secara in vitro sistem pembawa ubat koloid berasaskan Jintan Hitam untuk penghantaran merentasi halangan darah otak. Permulaannya, nanopartikel poli (D,L-laktik-ko asid glaikolik) (PLGA)/kitosan/Jintan Hitam telah difabrikasi melalui kaedah penyejatan-resapan pelarut yang telah dibangunkan dan pencirian dijalankan dari segi distribusi ukuran partikel, analisa cas dan morfologi permukaan. Dari ujian pencirian, lima nanopartikel yang telah difabrikasi telah dipilih mengikut saiz partikel dalam lingkungan 10 ke 970 nm dengan permukaan luar yang licin dan cas permukaan yang stabil untuk tujuan penyiasatan selanjutnya. Yang kedua, mikroemulsi minyak Jintan Hitam-dalam-air telah disediakan melalui titrasi titisan minyak Jintan Hitam dengan jumlah isipadu yang telah ditentukan lebih awal ke dalam campuran surfaktan (Span 20, Span 80, Tween 20, Tween 80, Tween 85) dan air. Semua campuran pertigaan yang telus telah dicirikan dari segi kelikatan, saiz partikel, kestabilan termodinamik, penampilan telus secara optikal dan kemampuan kelarutan yang tinggi. Kestabilan mikroemulsi telah dinilai dengan merujuk kepada keadaan tertekan iaitu pengemparan (2000 g selama 20 minit) dan pemanasan di dalam ketuhar pengering (60 to 105°C selama 5 jam) dan saiz titisan telah ditentukan selepas penyimpanan selama sebulan pada suhu bilik (25°C ± 2). Berdasarkan keputusan yang diperolehi, satu gambarajah fasa pertigaan telah dibina daripada jumlah ketiga-tiga komponen tersebut.

Campuran Jintan Hitam (dalam lingkungan 7.4 ke 10.7%) telah disediakan dari campuran surfaktan HLB 16 (Tween 20: Tween 80; 6:4) dengan air ternyahion (dalam lingkungan 17.9 ke 18.5%) untuk menghasilkan cecair yang telus. Gambarajah fasa yang dibina memaparkan kawasan-kawasan dari beberapa jenis mikroemulsi dan emulsi. Saiz titisan dari campuran yang baru disediakan adalah lebih luas (5 to 15.6 nm) berbanding saiz titisan selepas keadaan tertekan (11.3 to 12.4 nm). Data telah menunjukkan bahawa minyak Jintan Hitam boleh diformulasikan kepada mikroemulsi minyak-dalam-air dengan campuran surfaktan pada nilai HLB yang spesifik. Berikutan pencirian kedua-dua sistem pembawa ini, empat ujian in vitro telah dijalankan: (1) daya maju sel MDCK 1 dan N2a; (2) induksi pertumbuhan saraf pada sel N2a; (3) kecekapan transfeksi ke atas sel MDCK 1 dan N2a dan (4) ujian kebolehtelapan merentasi halangan darah otak berikutan pemuatan nanopartikel. Hasil kajian menunjukkan bahawa: (1) tiada ketoksikan sel yang signifikan didapati apabila sel-sel MDCK 1 dirawat dengan nanopartikel walaupun pada konsentrasi tertinggi, 1000 µg/ml. Mikroemulsi telah disimpulkan sebagai cukup kuat terhadap sel-sel N2a dan ia berfungsi dengan cara bergantung kepada dos; (2) saiz partikel yang kecil dan kecekapan pengkapsulan minyak Jintan Hitam yang tinggi boleh dikaitkan dengan kesan neuroregenerasi minyak Jintan Hitam dalam sel-sel N2a, yang membawa kepada pertumbuhan saraf; (3) ujian kecekapan transfeksi menunjukkan bahawa nanopartikel-kitosan dengan berat molekul sederhana memberikan transfeksi tertinggi ke dalam sel-sel. (4) Ujian kebolehtelapan menunjukkan bahawa nanopartikel-kitosan dengan berat molekul rendah memberikan nilai rintangan elektrik transepitelial (TEER) yang paling rendah, menandakan keupayaan penyerapan tertinggi berbanding nanopartikel yang lain.

Secara keseluruhan, sistem ini dijangka membolehkan ujian rutin in vitro ke atas sel saraf yang dimuatkan dengan minyak Jintan Hitam atau bahan lipofilik yang lain dapat dilakukan dengan cepat apabila pemerhatian ke atas pertumbuhan saraf diperlukan. Ia juga dicadangkan bahawa pembawa ubat koloid berasaskan Jintan Hitam akan membantu dalam kajian halangan darah otak pada masa hadapan menggunakan sistem penghantaran terapi gen bukan viral untuk rawatan penyakit neurodegenerasi.

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APPROVAL PAGE

I certify that I have supervised and read this study and that in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master of Pharmaceutical Science (Pharmaceutical Technology).

………..

Farahidah Mohamed Supervisor

………..

Mohd Affendi Mohd Shafri Co-supervisor

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master of Pharmaceutical Science (Pharmaceutical Technology).

.……….

Solachuddin Jauhari Arief Ichwan Internal Examiner

………..

Sabrina Sukardi External Examiner

This thesis was submitted to the Department of Pharmaceutical Technology and is accepted as a fulfilment of the requirement for the degree of Master of Pharmaceutical Science (Pharmaceutical Technology).

.……….

Juliana Md Jaffri Head, Department of Pharmaceutical Technology

This thesis was submitted to the Kulliyyah of Pharmacy and is accepted as a fulfilment of the requirement for the degree of Master of Pharmaceutical Science (Pharmaceutical Technology).

………..

Siti Hadijah Shamsudin Dean, Kulliyyah of Pharmacy

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DECLARATION

I hereby declare that this thesis is the result of my own investigations, except where otherwise stated. I also declare that it has not been previously or concurrently submitted as a whole for any other degrees at IIUM or other institutions.

Nurul Hafizah binti Mohd Nor

Signature………. Date …...

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vii

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH

Copyright ©2015 by Nurul Hafizah binti Mohd Nor. All rights reserved.

IN VITRO EVALUATION OF NIGELLA SATIVA-BASED COLLOIDAL DRUG CARRIERS

FOR DELIVERY ACROSS BLOOD-BRAIN BARRIER

No part of this unpublished research may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder except as provided below.

1. Any material contained in or derived from this unpublished research may be used by others in their writing with due acknowledgement.

2. IIUM or its library will have the right to make and transmit copies (print or electronic) for institutional and academic purposes.

3. The IIUM library will have the right to make, store in a retrieval system and supply copies of this unpublished research if requested by other universities and research libraries.

Affirmed by Nurul Hafizah binti Mohd Nor

……..……..……… ………..

Signature Date

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viii

…To my beloved mother, late father and siblings…

…The paradise of my heart…

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ACKNOWLEDGEMENTS

Alhamdulillahi rabbil ‘aalamin, peace and blessing be upon our beloved Prophet Muhammad S.A.W. First and foremost, my deepest gratitude to Allah S.W.T for His blessings and will in completing this research and thesis successfully.

An honorable gratitude goes to my mother, Hadazah Chik and all my siblings for their understandings and supports on me in completing this research. Without help from them, I would face many difficulties while doing this research.

A million thanks to my supervisor, Assoc. Prof. Dr. Farahidah Mohamed for the precious guidance and advice. Not only that, she continually and convincingly conveyed a spirit of adventure in regard to research and an excitement in regard to teaching. Besides, my deepest appreciation goes to my co-supervisor, Asst. Prof. Dr.

Mohd Affendi Mohd Shafri. He inspired me greatly to work in this research.

In addition, I would like to thank the science officer of Department of Pharmaceutical Technology, Sis. Zaililah and all laboratory assistants, Bro. Dzadil Syakirin, Bro. Tuan Faris, Bro. Amin and Sis Hazan Haryanti for their help and guidance as well as for providing me with good environments and facilities in completing this research.

The thank you also goes to all my project teammates, Bro. Abd Almonem and Nur „Izzati Mansor who provided me valuable information as the guidance of the research and had demonstrated to me about the techniques and procedures as well as willing to share the knowledge. Also, the gratitude also goes to Bro. Abdu Arrahman for being my place of reference when I was lost in statistical problems.

Finally, not to forget all my laboratory friends, Bro. Fahmi, Bro. Solahuddin, Fathin Athirah, Maryam Saadah, Huwaida, Putri Nur Hidayah, Tengku Faris and Anugerah for giving me assistance whenever I needed help, sharing the ideas and making my every day in the laboratory happy with their jokes. Also, special thanks to a best friend of mine for being there whenever I had problems and kept giving the spirit to be strong in the research field. May Allah shower you all with His blessing, insha Allah.

Thank you and salam.

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TABLE OF CONTENTS

Abstract ... ii

Abstract in Arabic ... iii

Abstract in Bahasa Melayu ... iv

Approval Page ... v

Declaration ... vi

Copyright Page ... vii

Dedication ... viii

Acknowledgements ... ix

List of Tables ... xiii

List of Figures ... xv

List of Equations ... xxii

List of Abbreviations ... xxiii

CHAPTER 1: INTRODUCTION ... 1

1.1 Introduction ... 1

1.2 Research Background ... 2

1.3 Literature Review ... 3

1.3.1 DNA-based Therapy ... 3

1.3.2 N. sativa Linn ... 6

1.3.3 Development of Nanoparticle System ... 9

1.3.3.1 Poly (D, L-lactide-co-glycolide) (PLGA) ... 10

1.3.3.1 Chitosan ... 11

1.3.4 Development of the Microemulsion System ... 13

1.3.4.1 An Overview ... 13

1.3.4.2 Classification of Microemulsions ... 14

1.3.5 Choices of Surfactants ... 15

1.3.5.1 An Overview ... 15

1.3.5.2 Classification of Surfactants ... 16

1.3.6 Neurite Outgrowth Study on N. sativa-based Colloidal System 18 1.3.7 In vitro Cell Cultures ... 20

1.3.7.1 Neuro-2a (N2a) Cells for Neurite Outgrowth Study ... 20

1.3.7.2 Blood-brain Barrier Model using MDCK 1 Cells ... 22

1.3.7.2.1 An Overview ... 22

1.3.7.2.2 Identifying Factors for Permeability ... 26

1.3.7.2.3 The Blood-brain Barrier Reconstructive Model29 1.4 Objectives and Scope of the Study ... 33

1.5 Hypotheses ... 33

CHAPTER 2: FORMULATIONS OF ‘NEUROBIONANOPARTICLES’ AND PREPARATION OF MICROEMULSIONS ... 34

2.1 Introduction ... 34

2.2 Materials and Chemicals ... 36

2.3 Methodology ... 37

2.3.1 Fabrication of pDNA-Loaded PLGA/Chitosan/N. sativa Nanoparticles ... 37

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xi

2.3.2 Characterization of pDNA-Loaded PLGA/Chitosan/N. sativa

Nanoparticles ... 40

2.3.2.1 Determination of Particle Size Distribution ... 40

2.3.2.2 Determination of Surface Charge ... 40

2.3.2.3 Observation of Surface Morphology using Field Emission Scanning Electron Microscopy (FESEM) .... 41

2.3.2.4 Transfection Efficiency on N2a Cells ... 41

2.3.3 Preparation of the N. sativa Oil-in-Water Microemulsions ... 42

2.3.3.1 HLB Preparation ... 42

2.3.3.2 Preparation of N. sativa Oil-in-Water Microemulsions 44 2.3.4 Characterization of N. sativa Oil-in-Water Microemulsions .... 47

2.3.4.1 Determination of Particle Size Distribution ... 47

2.3.4.2 Determination of Viscosity ... 47

2.3.4.3 Stress-tests and Stability Study ... 47

2.3.5 Construction of Ternary Phase Diagram ... 48

2.3.6 Statistical Analysis ... 48

2.4 Results and Discussion ... 49

2.4.1 Fabrication of pDNA-loaded PLGA/Chitosan/N. sativa Nanoparticles ... 49

2.4.2 Characterization of pDNA-loaded PLGA/Chitosan/N. sativa Nanoparticles ... 52

2.4.2.1 Particle Size Distribution, Surface Morphology and Charge Analysis ... 52

2.4.3 Transfection Efficiency on N2a Cells ... 61

2.4.4 Preparation of N. sativa Oil-in-Water Microemulsions ... 63

2.4.5 Characterization of N. sativa Oil-in-Water Microemulsions .... 68

2.4.5.1 Droplet Size and Viscosity ... 68

2.4.5.2 Stress-tests and Stability of the N. sativa Oil-in-Water Microemulsions ... 69

2.4.6 Phase Behavior of N. sativa Oil-in-Water Microemulsions ... 73

2.5 Conclusion ... 76

CHAPTER 3: COMPARISON OF NEURITE OUTGROWTH STUDY OF pDNA-LOADED PLGA/CHITOSAN/N. SATIVA NANOPARTICLES AND MICROEMULSIONS USING NEURO-2A MURINE NEUROBLASTOMA CELLS ... 77

3.1 Introduction ... 77

3.2 Materials and Chemicals ... 78

3.3 Methodology ... 78

3.3.1 N2a Cell Culture and Maintenance ... 78

3.3.2 Cell Counting and Seeding ... 79

3.3.3 Cell Viability Study of N2a Cells ... 79

3.3.4 Neurite Outgrowth Study ... 82

3.3.5 Measurement of Neurite Length ... 85

3.3.6 Statistical Analysis ... 85

3.4 Results and Discussion ... 86

3.4.1 Cell Viability Study ... 86

3.4.2 Neurite Outgrowth Study ... 90

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3.4.3 Comparison of the Neurite Outgrowth Induction between

Nanoparticles and Microemulsions in N2a Cells ... 111

3.4.4 Mechanisms of Actions of Neurite Outgrowth in N2a Cells .. 111

3.5 Conclusion ... 114

CHAPTER 4: PERMEABILITY STUDY OF pDNA-LOADED PLGA/CHITOSAN/N. SATIVA NANOPARTICLES USING MDCK 1 CELL LINE ... 115

4.1 Introduction ... 115

4.2 Materials and Chemicals ... 116

4.3 Methodology ... 117

4.3.1 MDCK 1 Cell Culture and Maintenance ... 117

4.3.2 Cell Viability of MDCK 1 Cells ... 117

4.3.3 Modelling in vitro Blood-brain Barrier ... 117

4.3.4 TransEpithelial Electrical Resistance (TEER) Measurements . 120 4.3.5 Transfection Efficiency of MDCK 1 Cells ... 123

4.3.6 Statistical Analysis ... 123

4.4 Results and Discussion ... 123

4.4.1 Cell Viability of MDCK 1 Cells ... 123

4.4.2 Blood-Brain Barrier Modelling ... 125

4.4.2.1 MDCK 1 Cells and Target Proteins of Tight Junctions 126 4.4.3 Permeability Study of Blood-Brain Barrier ... 128

4.4.4 Effects of Chitosan on the Opening of the Tight Junctions ... 134

4.4.5 Effect of Particle Size on the Modulation of Tight Junction ... 137

4.4.6 Effect of Polysorbate Surfactant ... 138

4.4.7 Effect of the N. sativa Oil ... 139

4.4.8 Recovery of the Tight Junctions ... 139

4.4.9 Transfection Efficiency of MDCK 1 Cells ... 140

4.5 Conclusion ... 144

CHAPTER 5: GENERAL DISCUSSIONS ... 145

CHAPTER 6: CONCLUSION ... 150

6.1 Future Study ... 150

REFERENCES ... 152

APPENDIX I: PUBLICATIONS AND PRESENTATIONS ... 166

APPENDIX II: CERTIFICATE OF ANALYSIS ... 167

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xiii

LIST OF TABLES

Table No. Page No.

1.1 Description of the plasmid map of pMCS-Gaussia Luc

Vector (Thermo Scientific literature, 2014) 5

2.1 The various ingredients tested in the fabrication of the nanoparticles. For all formulations; 9 ml of the total volume was used in the aqueous phase, whereas 36 ml of the

dispersion media was used in the hardening tank 39 2.2 The HLB values of the surfactants used in the preparation of

the N. sativa oil-in-water microemulsions. These different surfactants were formulated to produce the HLB blends with

hydrophilic properties 44

2.3 Preparations of formulations made up of various surfactant

types, blends and different HLB values 46

2.4 The ingredients of the successfully fabricated pDNA-loaded

PLGA/Chitosan/N. sativa nanoparticles 50

2.5 Different types of chitosan used in the nanoparticles

fabrication 52

2.6 Particle size of the non-lyophilized and lyophilized nanoparticle groups suspended in various suspension media.

There were significant differences in the mean particle size of the nanoparticle groups as compared to the control group (One-Way ANOVA, Dunnet‟s Test, p = 0.00). Different letter indicated significant difference in the mean values

among nanoparticle groups 53

2.7 The physical appearances of the microemulsions with

different surfactant blends and HLB values 67

2.8 Physical characteristics of N. sativa oil-in-water microemulsions. The asterisk (*) represented the formulation groups that were significantly different (One-Way ANOVA, post-hoc Tukey‟s test multiple comparison, p < 0.05)

68 2.9 Stress-tests for phase separation of N. sativa oil-in-water

microemulsions 69

2.10 The turbidity changes of the N. sativa oil-in-water

microemulsions after one-month storage 72

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Table No. Page No.

3.1 Different concentrations of pDNA-loaded PLGA/chitosan/N.

sativa nanoparticles and N. sativa oil-in-water

microemulsions were tested for cell viability assay 80 3.2 All formulations were tested based on the tabulated

concentrations for neurite outgrowth study 83

3.3 IC50 values of N2a cells loaded with different concentrations

of N. sativa oil-in-water microemulsions 89

3.4 Measurements of the neurite outgrowth of nanoparticles at various concentrations. Some values have high standard deviation due to the presence of some cells having an unusual value in neuron length. There were significant differences in neurite lengths among the various concentrations of nanoparticle groups compared to Control

NP (One-Way ANOVA, Dunnet‟s test, p = 0. 00) 92 3.5 The encapsulation efficiency of the N. sativa oil

encapsulated in the pDNA-loaded PLGA/chitosan/N. sativa

nanoparticles 93

3.6 Measurement of neurite extension of N2a cells loaded with various microemulsions at 24, 48, 72 and 96 hours post- incubation. There were significant differences among the concentrations in each microemulsion formulation (One- Way ANOVA, Tukey‟s test, p < 0.05)

100 3.7 Measurement of neurite extension of the untreated N2a cells

in serum-free media and cells loaded with various concentrations of the controls; dbcAMP, Tween 20 and

Tween 80 at 24, 48, 72 and 96 hours post-incubation 105 4.1 Comparison of the zeta potential measurements between the

non-chitosan bearing nanoparticles vs NBP (1) and NBP (2) bearing low molecular weight and medium molecular weight

of chitosan, respectively 135

4.2 Characteristics of the chitosan used in the preparation of the nanoparticles. The degree of deacetylation and molecular weight were important factors contributing to the modulation of the tight junction and the transfection efficiency of

MDCK 1 cells 136

4.3 The amount of pDNA transfected equivalent to the

percentage of the in vitro release 142

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LIST OF FIGURES

Figure No. Page No.

1.1 Schematic diagram of the pMCS-Gaussia Luc Vector encoding the Gaussia luciferase reporter (Thermo Scientific

literature, 2014). 5

1.2 Picture of N. sativa plant. The flowers are delicate and usually colored in pale blue and white with five to ten petals

(Get Well Natural LLC literature, n.d). 7

1.3 Structure of PLGA synthesized by the process of random- ring opening co-polymerization of glycolic acid linked by

ester bond (Bouissou & Walle, 2006). 10

1.4 Structure of chitosan synthesized through the N-

deacetylation of chitin (Guolin et al., 2012). 12 1.5 Three different classifications of the microemulsion systems

which are; Type I (oil-in-water microemulsion), Type II (water-in-oil microemulsion) and Type III (bicontinuous

microemulsion) (Winsor, 1948). 15

1.6 Schematic picture of neuron cell showing two different parts; dendrites and axon forming the neurites (Bear et al.,

2006). 20

1.7 Morphology of normal N2a cells at 90% confluency cultured in complete DMEM. Red arrow shows one of the

cells with amoeboidal shape. 21

1.8 Cellular components of the blood-brain barrier (Wilhelm,

Fazakas & Krizbai, 2011). 23

1.9 The mechanism of the transport across the cells (King,

2011). 25

1.10 Tight junction proteins of the epithelial cells. It is reported to have similar tight junctions as in the endothelial blood-

brain barrier cells (Qiagen Company literature, 2014). 28 2.1 Schematic diagram of the fabrication of pDNA-loaded

PLGA/chitosan/N. sativa nanoparticles using the diffusion-

solvent-evaporation method. 38

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Figure No. Page No.

2.2 Transfection efficiency of N2a loaded with different nanoparticle formulations equivalent to 20 µg of the

encapsulated pDNA. 42

2.3 The working HLB scale of the surfactants (ICI Americas,

1976). 43

2.4 A flowchart to describe the preparation of the N. sativa oil-

in-water microemulsions. 45

2.5 The postulated schematic diagram of the fabricated

nanoparticles showing three different phases. 49 2.6 Images of nanoparticles obtained from Field Emission

Scanning Electron Microscopy (FESEM). The nanoparticles showed smooth exterior surfaces with spherical-shaped with

particle size in the range of 10 to 200 nm. 55 2.7 The adsorption of the positively charged surface coating on

the negatively charged nanoparticle. 57

2.8 Zeta potential values of the non-lyophilized nanoparticles suspended in the deionized water. Data represented by means ± SEM. The group with asterisk (*) represented significant difference compared to the control group (One-

Way ANOVA, Dunnet‟s test, p < 0.05). 58

2.9 Zeta potential values of the lyophilized nanoparticles suspended in the deionized water. Data represented by means ± SEM. The group with asterisk (*) represented significant difference compared to the control group (One-

Way ANOVA, Dunnet‟s test, p < 0.05). 59

2.10 Zeta potential values of the lyophilized nanoparticles suspended in the HBSS. Data were represented by means ±

SEM. 59

2.11 The transfection efficiency of N2a cells loaded with various nanoparticle groups equivalent to 20 µg pDNA. The measurements were taken from triplicates studies (n=3) and the results were shown as means ± SEM. Means that do not

share a letter were significantly different. 61 2.12 Schematic diagram of Type I (oil-in-water microemulsion).

The oil phase is dispersed throughout the water phase in

separated droplets. 64

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Figure No. Page No.

2.13 Physical appearances of the transparent N. sativa oil-in-

water microemulsions. 66

2.14 The triplicate measurements of the zeta potential of all microemulsion formulations. The results showed decrement of the zeta potential reading of the after one month storage

at room temperature (25ºC ± 2). Note. F = Formulation. 71 2.15 Pseudoternary diagram of the N. sativa oil-in-water

microemulsions. Formulation 14, 15 and 16 were closely spotted near to the upper left of the phase region, indicating that these formulations were the N. sativa oil-in-water microemulsions.

74 2.16 Hypothetical phase region of microemulsion systems of oil,

water and surfactant systems. 74

3.1 Flow chart of cell viability assay of N2a cells. 81 3.2 Flow chart of neurite outgrowth study loaded with different

concentrations of pDNA-loaded PLGA/chitosan/N. sativa nanoparticles and N. sativa oil-in-water microemulsions.

The neurite outgrowth was observed at 24 hours, 48 hours, 72 hours and 96 hours post-treatment using the inverted

microscopy. 84

3.3 Cell viability of N2a loaded with nanoparticles at various concentrations. The absorbance measurements were taken at 48 hours post incubation. All samples did not reach 50% of the inhibition/lethal concentration of the total cells, suggested being safe and gave no cytotoxic effect towards

N2a cells. 87

3.4 In vitro cell viability of N2a cells loaded with different concentrations of microemulsions. All formulations did reach 50% of the inhibition/lethal concentration of the total

cells. 88

3.5 Cell viability study of N2a loaded with different concentrations of dbcAMP. The dbcAMP was observed to block the cytotoxicity activity in concentration-dependent

manner. 90

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xviii

Figure No. Page No.

3.6 Neurite outgrowth of N2a cells. A(i) represented 100 µg/ml of Control NP incubated for 24 hours. A(ii) represented 100 µg/ml of Control NP incubated for 96 hours. B(i) represented 100 µg/ml of NP (A) incubated for 24 hours.

B(ii) represented 100 µg/ml of NP (A) incubated for 96 hours. At 96 hours post-incubation, cells treated with Control NP gave little extension. Cells treated with NP (A) looked isolated from each other, differentiated and survived.

Red bar represented 20 µm. Red arrows indicated the

neurite protrusion. 96

3.7 Neurite outgrowth of N2a cells. C(i) represented 100 µg/ml of NP (B) incubated for 24 hours. C(ii) represented 100 µg/ml of NP (B) incubated for 96 hours. D(i) represented 100 µg/ml of NBP (1) incubated for 24 hours. D(ii) represented 100 µg/ml of NBP (1) incubated for 96 hours.

At 96 hours post-incubation, cells looked isolated from each other, differentiated and survived. Red bar represented 20

µm. Red arrows indicated the neurite protrusion. 97 3.8 Neurite outgrowth of N2a cells. E(i) represented 100 µg/ml

of NBP (2) incubated for 24 hours. E(ii) represented 100 µg/ml of NBP (2) incubated for 96 hours. At 96 hours post- incubation, cells looked isolated from each other, differentiated and survived. Red bar represented 20 µm. Red

arrows indicated the neurite protrusion. 98

3.9 Neurite outgrowth of N2a cells. F(i) represented 0.5 µl/ml of Formulation 14 at 24 hours post-incubation. F(ii) represented 0.5 µl/ml of Formulation 14 at 96 hours post- incubation. G(i) represented 0.5 µl/ml of Formulation 15 at 24 hours post-incubation. G(ii) represented 0.5 µl/ml of Formulation 15 at 96 hours post-incubation. At 96 hours post-incubation, cells looked healthy, isolated and differentiated. Red bar represented 20 µm. Red arrows

indicated the neurite protrusion. 102

3.10 Neurite outgrowth of N2a cells. H(i) represented 0.5 µl/ml of Formulation 16 at 24 hours post-incubation. H(ii) represented 0.5 µl/ml of Formulation 16 at 96 hours post- incubation. At 96 hours post-incubation, cells looked healthy, isolated and differentiated. Red bar represented 20

µm. Red arrows indicated the neurite protrusion. 103

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Figure No. Page No.

3.11 Neurite outgrowth of N2a cells. I(i) loaded with 500 µg/ml of dbcAMP incubated at 24 hours post-incubation. I(ii) loaded with 500 µg/ml of dbcAMP incubated at 96 hours post-incubation. J(i) loaded with 60% v/v Tween 20 incubated at 24 hours post-incubation. J(ii) loaded with 60%

v/v Tween 20 incubated at 96 hours post-incubation. At 96 hours post-incubation, cells incubated with dbcAMP showed the differention. The cells loaded with Tween 20 looked shrank and dispersed to certain areas. Red bar

represented 20 µm. 107

3.12 Neurite outgrowth of N2a cells. K(i) loaded with 40% v/v Tween 80 incubated at 24 hours post-incubation. K(ii) loaded with 40% v/v Tween 80 incubated at 96 hours post- incubation. L(i) untreated N2a cells in serum-free media at 24 hours post-incubation. L(ii) untreated N2a cells in serum- free media at 96 hours post-incubation media. At 96 hours post-incubation, cells in serum-free media and cells loaded with Tween 80 looked shrank and dispersed to certain areas.

Red bar represented 20 µm. 108

3.13 Neurite outgrowth of N2a cells. M(i) untreated cells in complete growth media incubated at 24 hours post- incubation. M(ii) untreated cells in complete growth media incubated at 96 hours post-incubation. At 96 hours post- incubation, cells looked rounded, proliferated and

undifferentiated. Red bar represented 20 µm. 110 4.1 Protocol of the permeability assay of blood-brain barrier

model using MDCK 1 cells modified from Hombach &

Bernkop-Schnürch (2009). 118

4.2 The in vitro blood-brain barrier model using MDCK 1 cells.

The cells were plated on 0. 4 μm PET filter of the Millipore hanging cell culture inserts on the 24-well plates supplemented with 0.3 ml of the complete growth medium in the luminal space and 1.1 ml of the complete growth medium in the abluminal space. The volume of the media in the insert and in the well should be at the same level to avoid hydrostatic pressure on the membrane. For each concentration, the experiment was performed in triplicate of

inserts (n=3). 120

4.3 The electrodes with different lengths were inserted in the wells. The shorter electrode was placed in the insert without touching the filter membrane, and another longer electrode

was inserted in the well. 121

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xx

Figure No. Page No.

4.4 In vitro cell viability of MDCK 1 cells loaded with different concentrations of nanoparticles. There was significant difference in the cell viability among the various concentrations of nanoparticles (One-Way ANOVA, post-

hoc Tukey‟s test, p < 0. 05). 124

4.5 Naked eyes observation of MDCK 1 cells treated with NBP (2). Each concentration of NBP (2) were loaded in triplicate wells (n=3) at 48 hours post-treatment. The violet formazan formed indicated that the neurobionanoparticle did not give

significant toxicity to the cells. 125

4.6 Schematic diagram of the in vitro blood-brain barrier model with EOO culture arrangement. Only epithelial cells (E) were plated onto the well of the insert in the luminal space.

The first (O) was referring to the absent cells on the bottom of the porous membrane and the later (O) referred to the absent cells on the bottom of the wells in the abluminal

space. 126

4.7 Single confocal plane of tight junction markers of Madin- Darby Canine Kidney (MDCK) cells. The white arrows indicated the respective (a) ZO-1 and (b) occludin of the

MDCK cells (Cheung et al., 2011). 127

4.8 The structure of endothelial and epithelial tight junction showing central importance of claudin, ZO-1 and actin. The similarity in the presence of the tight junction proteins made the using of the epithelial cells in modelling the blood-brain

barrier. 127

4.9 TEER measurement (n=3) of MDCK 1 cell monolayer loaded with various concentrations of Control NP at different intervals. The results were shown as means ± SEM. The groups with asterisk (*) represented the significant difference compared to the control group (One-

Way ANOVA, post-hoc Dunnet‟s test, p < 0.05). 129 4.10 TEER measurement (n=3) of MDCK 1 cell monolayer

loaded with various concentrations of NP (A) at different intervals. The cells supplied with HBSS alone served as

control. The results were shown as means ± SEM. 130

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Figure No. Page No.

4.11 TEER measurement (n=3) of MDCK 1 cell monolayer loaded with various concentrations of NP (B) at different intervals. The cells supplied with HBSS alone served as control. The results were shown as means ± SEM. The groups with asterisk (*) represented the significant difference compared to the control group (One-Way

ANOVA, post-hoc Dunnet‟s test, p < 0.05). 130 4.12 TEER measurement (n=3) of MDCK 1 cell monolayer

loaded with various concentrations of NBP (1) at different intervals. The cells supplied with HBSS alone served as control. The results were shown as means ± SEM. The groups with asterisk (*) represented the significant difference compared to the control group (One-Way

ANOVA, post-hoc Dunnet‟s test, p < 0.05). 131 4.13 TEER measurement (n=3) of MDCK 1 cell loaded with

various concentrations of NBP (2) at different intervals.

The results were shown as means ± SEM. The groups with asterisk (*) represented the significant difference compared to the control group (One-Way ANOVA, post-hoc Dunnet‟s

test, p < 0.05). 131

4.14 The effect of 350 µg/well of various nanoparticles towards the integrity of MDCK 1 cell monolayer following time dependent manner permeability assay (0 minute to 24 hours). The TEER measurements were taken from triplicates studies (n=3) and the results were shown as means ± SEM. The groups with asterisk (*) represented significant difference in TEER measurements among the various nanoparticles as compared to the control group

(One-Way ANOVA, post-hoc Dunnet‟s test, p < 0.05). 133 4.15 The structures of positively charged chitosan. The red oval

shape indicated the protonated amines, having pKa of ± 6.2

was suggested to cause the positively charge of the chitosan. 135 4.16 Transfection efficiency of MDCK 1 cell line transfected

with different nanoparticles at various concentrations. The groups with asterisk (*) were significantly different compared to the positive control level mean (One-Way

ANOVA, post-hoc Dunnet‟s test, p < 0.05). 141 4.17 Transfection efficiency of MDCK 1 cells loaded with 350

µg/well of various nanoparticle groups. All groups were not significantly different compared to the positive control level

mean. Data were represented by means ± SEM. 143

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LIST OF EQUATIONS

Equation No. Page No.

2.1 43

4.1 122

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xxiii

LIST OF ABBREVIATIONS

ATCC American Type Culture Collection cGMP Guanosine 3′, 5′-cyclic MonoPhosphate

CNS Central Nervous System

CO2 Carbon Dioxide

CS Chitosan

CTAB CetylTrimethylAmmonium Bromide

dbcAMP Dibutryl Cyclic Adenosine MonoPhosphate

DMEM Dulbecco‟s Minimum Essential Medium

DMSO Dimethyl sulfoxide

EDTA EthyleneDiamineTetraacetic Acid

EE Encapsulation Efficiency

EGFR Epidermal Growth Factor Receptor EVOM2 Epithelial Voltohmmeter2

FBS Fetal Bovine Serum

FESEM Field Emission Scanning Electron Microscopy

GLA Gamma Linolenic Acid

HBSS Hanks Balanced Salt Solution

HIV Human Immunodeficiency Virus

HLB Hydrophile-Lipophile Balance

IC50 Half Maximal of Inhibitory Concentration JACOP Junction-Associated Coiled-Coil Protein MDCK 1 Madin-Darby Canine Kidney 1 Cell Line MEK Mitogen-activated protein kinase/ERK kinase

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MPS Mononuclear Phagocyte System

MTT 3-(4, 5-Dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide N2a Neuro-2a Murine Neuroblastoma Cell Line

NaOH Natrium Hydroxide

NOS Nitric Oxide Synthase

PBS Phosphate Buffered Saline

PCL Poly (CaproLactone)

PDI PolyDispersity Index

pDNA Plasmid Deoxyribonucleic Acid

PDZ Postsynaptic density 95, PSD-85; Drosophila discs large protein, Dlg; Zonula occludens-1, ZO-1)

PET PolyEthylene Terephthalate

P-gp P-glycoprotein

PLA Poly Lactic Acid

PLGA Poly (D, L-Lactide-co-Glycolic Acid) pMCS Plasmid Multiple Cloning Sites

PVA Poly (Vinil Alcohol)

RTK Receptor Tyrosine Kinases

siRNA Small Interfering Ribonucleic Acid

SLC Solute Carrier

TEER TransEpithelial Electrical Resistance

VE Vascular Endothelial

ZO-1 Zonula Occludin-1

ZO-1 Zonula Occludin-2

Rujukan

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