DEVELOPMENT of a THERAPEUTIC MODEL of EARLY LIVER CANCER USING CROCIN-COATED MAGNETITE NANOPARTICLES Rkia El-Kharrag
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United Arab Emirates University Scholarworks@UAEU Dissertations Electronic Theses and Dissertations 4-2015 DEVELOPMENT OF A THERAPEUTIC MODEL OF EARLY LIVER CANCER USING CROCIN-COATED MAGNETITE NANOPARTICLES Rkia El-Kharrag Follow this and additional works at: https://scholarworks.uaeu.ac.ae/all_dissertations Part of the Biology Commons Recommended Citation El-Kharrag, Rkia, "DEVELOPMENT OF A THERAPEUTIC MODEL OF EARLY LIVER CANCER USING CROCIN-COATED MAGNETITE NANOPARTICLES" (2015). Dissertations. 14. https://scholarworks.uaeu.ac.ae/all_dissertations/14 This Dissertation is brought to you for free and open access by the Electronic Theses and Dissertations at Scholarworks@UAEU. It has been accepted for inclusion in Dissertations by an authorized administrator of Scholarworks@UAEU. For more information, please contact [email protected]. i United Arab Emirates University College of Science DEVELOPMENT OF A THERAPEUTIC MODEL OF EARLY LIVER CANCER USING CROCIN-COATED MAGNETITE NANOPARTICLES Rkia El-kharrag This dissertation is submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy Under the Supervision of Professor Amr Amin April 2015 ii Declaration of Original Work I, Rkia El-Kharrag, the undersigned, a graduate student at the United Arab Emirates University (UAEU), and the author of this PhD dissertation, entitled “Development of a Therapeutic Model of Early Liver Cancer Using Crocin-Coated Magnetite Nanoparticles”, hereby, solemnly declare that this dissertation is an original research work that has been done and prepared by me under the supervision of Dr. Amr Amin, in the College of Science at the UAEU. This work has not been previously formed as the basis for the award of any academic degree, diploma or a similar title at this or any other university. The materials borrowed from other sources and included in my dissertation have been properly cited and acknowledged. Student’s Signature Date ______________________ iii Copyright ©2015 by Rkia El-Kharrag All Rights Reserved iv Approval of the Doctorate Dissertation This Doctorate Dissertation is approved by the following Examining Committee Members: 1) Advisor (Committee Chair): Amr Amin Title: Professor Department of Biology College of Science Signature Date 2) Member: Yaser Greish Title: Associate Professor Department of Chemistry College of Science Signature Date 3) Member: Sherif Karam Title: Professor Department of Anatomy College of Medicine and Health Sciences Signature Date 4) Member (Internal Examiner): Ernest Adeghate Title: Professor Department of Anatomy College of Medicine and Health Sciences Signature Date 5) Member (External Examiner): Lily Yang Title: Professor College: School of Medicine, Department of Surgery Institution: Emory University, Atlanta, USA Signature Date v This Doctorate Dissertation is accepted by: 1) Dean, College of Science: Professor Frederick Chi-Ching Leung Signature: Date: 2) Dean, College of the Graduate Studies: Professor Nagi T. Wakim Signature: Date: Copy _____ of _____ vi Abstract Hepatocellular carcinoma is one of the most common health problems that is difficult to treat. As a result of the side effects frequently experienced with conventional cancer treatments, there has been a growing interest to develop controlled drug delivery system that can reduce the mortality rate of liver cancer patients and un- harm healthy tissues. Magnetite nanoparticles are potentially important in hepatocellular carcinoma treatment, since they can be used as delivery system. Pure and coated magnetite nanoparticles were synthesized via modified co-precipitation method in air at low temperature. Various reaction parameters and coating materials have been investigated and characterized. Among these parameters and coating materials, 1.0 % of dextran was selected as an optimum coating for nanoparticles using a slow feeding rate for the Fe2+/Fe3+ reactants, maintaining the stirring and soaking temperatures at 60ºC. After that dextran-coated magnetite nanoparticles were bound to crocin, a pharmacologically active component of saffron, via cross-linker. Crocin alone has shown anti-cancer activity in different in vitro and in vivo settings by several studies. The aim of this study was to synthesize dextran-coated magnetite nanoparticles containing crocin with a higher therapeutic index for hepatocellular carcinoma treatment. The nanoparticles with crocin were tested in vitro and in vivo for their anti-cancer effects as compared to free crocin. HepG2 cells treated with crocin-dextran-coated magnetite nanoparticles showed a decrease in cell proliferation compared to control (non-treated cells) or to those treated with free crocin or dextran-coated nanoparticles. The anti-cancer activity of crocin-dextran-coated nanoparticles was also evaluated in Balb/c mice. These mice were injected with carcinogenic agent, diethylnitrosamine. Histological examination revealed several vii precancerous changes. The immunohistochemical analysis using antibodies indication of cell proliferation (Ki-67), apoptosis (M30-Cytodeath and Bcl-2), inflammation (cyclooxygenase-2) and angiogenesis (vascular endothelial growth factor), indicated that magnetite nanoparticles conjugated with dextran plus crocin does indeed improve its anti-tumorigenic activity over free crocin. These results provide the basis for designing new modalities for treatment of liver cancer which could hopefully reduce its high mortality rate. Keywords: Magnetite nanoparticles, dextran, precancerous changes, crocin, diethylnitrosamine, immunohistochemical studies. viii Title and Abstract (in Arabic) إنشاء نموذج لعﻻج سرطان الكبد المبكر باستخدام جزيئات النانو الممغنطة و المغلفة بالكروسين )oicorc( الملخص يعتبر سرطان الكبد من اكثر المشاكل الصحية شيوعا التي يصعب عﻻجها. ونتيجة ﻵثار الجانبية التي عادة ما تصاحب وسائل العﻻج التقليدية للسرطان, فقد كان هناك اهتمام متزايد لتطوير نظام التحكم في توجيه وتوصيل اﻻدوية إلى مكان السرطان التي يمكن أن تقلل من معدل الوفيات بسرطان الكبد وﻻ تحدث ضرر باﻷنسجة السليمة. جزئيات النانو الممغنطة يمكن أن تكون مهمة لعﻻج سرطان الكبد لما لها من دور في زيادة كفاءة توجيه وتوصيل اﻷدوية. تم تحضير جزئيات النانو الممغنطة النقية والمغلفة بطريقة خاصة ومعدلة في الهواء وفي درجة حرارة منخفضة. كما تم دراسة مختلف عوامل ومعايير التفاعل ومواد التغليف. ومن بين العديد من تلك المعايير ومواد التغليف, فقد اختير 1.0% من الديكستران )nartxed( كالتلغليف اﻷمثل لجزيئات النانو باستخدام معدل تنزيل بطيء للحديد الثنائي والثﻻثي مع الحفاظ على درجات حرارة التحريك والتنقيع في 60 درجة مئوية. وبعد ذلك تم تحضير جزيئات النانو الممغنطة والمغلفة بالديكستران و بالكروسين. الكروسين هو العقار النشط في نبات الزعفران. وقد أثبت الكروسين عند استخدامه بمفرده قدرته في مكافحة السرطان في مختلف التجارب المخبرية بواسطة العديد من الدراسات. وكان الهدف من هذه الدراسة هو انتاج جزيئات النانو الممغنطة و المغلفة بالديكستران والتي تحتوي على الكروسين ودراسة قدرتها العﻻجية لسرطان الكبد حيث تم اختبار قدرة جزيئات النانو مع الكروسين في المختبر. ولقد تم اختبار قدرة جزيئات النانو مع الكروسين في المختبر على مكافحة السرطان. و لوحظ أن الخﻻيا السرطانية للكبد )HepG2( تراجعت في تكاثرها بعد معالجتها بجزيئات النانو المغلفة بالديكستران و الكروسين مقارنة مع الخﻻيا الغير معالجة, أو الخﻻيا المعالجة بالكروسين بمفرده أو بجزيئات النانو المغلفة بالديكستران وبدون الكروسين. كما تم تقييم قدرة جزيئات النانو المغلفة بالديكستران و الكروسين على مكافحة السرطان في فئران التجارب )Balb/c(. حيث تم حقنها بمادة مسرطنة )diethylnitrosamine) . وكشف الفحص النسيجي على تواجد العديد من التغيرات المصاحبة للمراحل اﻷولى من اﻻصابه بالسرطان. كما أشار التحليل الكيمياء النسيجي المناعي )immunohistochemical( باستخدام اﻷجسام المضادة المعنية بالتعرف على ix عامل التكاثر الخلوي )Ki-67(, وعامل موت الخﻻيا المبرمج )M30-Cytodeath and Bcl-2( , وعامل اﻻلتهابات الخلوية (cyclooxygenase-2) وعامل نمو اﻷوعية الدموية الجديدة ) vascular endothelial growth factor( إلى أن جزيئات النانو المغلفة بالديكستران والكروسين أظهرت تحسنا ملموسا في مكافحة السرطان مقارنة بالكروسين بمفرده. تمثل تلك النتائج اﻷساس لتصميم طرق جديدة لعﻻج سرطان الكبد الذي نأمل في أن يساهم في خفض معدﻻت الوفيات المرتفعة لهذا المرض العضال. الكلمات الرئيسية: جزيئات النانو الممغنطة, ديكستران, التغيرات المصاحبة للمراحل اﻷولى من اﻻصابة بالسرطان, كروسين, المادة المسرطنة diethylnitrosamine, دراسات التحليل الكيميائي النسيجي المناعي. x Acknowledgments First and foremost I thank Allah for giving me strength and ability to accomplish my Ph.D. degree. I am thankful to the UAE University for giving me this opportunity and providing me with scholarship and funding my research. I am grateful to my supervisor Dr. Amr Amin for his guidance, assistance and encouragement during the years of my project. It has been an honour to be his first Ph.D. student. My sincere appreciation to Dr. Yaser Afifi and Dr. Sherif Karam for giving me the opportunity to gain from their experience and knowledge, use their laboratories, as well as for their support and confidence in me. They both encouraged me to explore new ideas, answer my questions and never failed to support me and advice in times where I felt uncertain despite being very busy. In addition to academic knowledge, they taught me to maintain a positive attitude and passion for science and research. As Dr. Yaser always says” Be optimistic and look at the filled half of the cup, not at the empty one”. If I ever have given a chance, I think Dr. Yaser’s and Dr. Sherif’s names deserve to be in the front page of my dissertation, because without their help throughout the years of my Ph.D. I could not achieve what I achieved today. I would like to thank the laboratory members of Dr. Yaser and Dr. Sheirf. These