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Detection of Folate Receptor from FR+ Cells Gopikrishna Pinnaka Governors State University

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Detection of Folate Receptor from FR+ Cells Gopikrishna Pinnaka Governors State University Governors State University OPUS Open Portal to University Scholarship All Capstone Projects Student Capstone Projects Spring 2012 Detection of Folate Receptor from FR+ Cells Gopikrishna Pinnaka Governors State University Follow this and additional works at: http://opus.govst.edu/capstones Part of the Analytical Chemistry Commons Recommended Citation Pinnaka, Gopikrishna, "Detection of Folate Receptor from FR+ Cells" (2012). All Capstone Projects. 11. http://opus.govst.edu/capstones/11 For more information about the academic degree, extended learning, and certificate programs of Governors State University, go to http://www.govst.edu/Academics/Degree_Programs_and_Certifications/ Visit the Governors State Analytical Chemistry Department This Project Summary is brought to you for free and open access by the Student Capstone Projects at OPUS Open Portal to University Scholarship. It has been accepted for inclusion in All Capstone Projects by an authorized administrator of OPUS Open Portal to University Scholarship. For more information, please contact [email protected]. Detection of Folate Receptor from FR+ Cells A Project Submitted To Governors State University By Gopikrishna Pinnaka In Partial Fulfillment of the Requirements for the Degree Of Masters in Science May 2012 Governors State University University Park, Illinois 1 Dedicated to My Parents, Wife & Friends 2 Acknowledgement: I would like to take this opportunity to offer my sincere thanks to each and every person for helping me to accomplish this task . This research project would not have been possible without the support of many people. I would like to express my gratitude thank to Dr. Walter Henne who was abundantly helpful and offered invaluable assistance, guidance and support throughout my project, without whose knowledge and assistance this study would not have been successful. I would like to thank Prof Kent and Dr. Addison for accepting to be as a committee members and providing me excellent skills in chromatography. Special thanks also to all my graduate friends, especially my best friend Audrea Rhymes for invaluable assistance. It was great to work with him, he always been there for me. I am very appreciative to many efforts on Dr. Henne’s behalf to work around my work schedule, and worked with me after hours to get things done. I am indebted to many student colleagues for providing stimulating and fun environment. I am especially grateful to all my friends and project partnersVikram Reddy, Audrea Rhymes and Naresh Reddy Vemula for helping me get through the difficult times for all the emotional support, company, entertainment provided. I wish to express my love and gratitude to beloved Parents, Brothers and my wife Priyanka for their understanding & endless love, through the duration of my studies. 3 Table of Contents: Abstract: ............................................................................................................................ 5 Introduction: ..................................................................................................................... 6 Cancer therapy: ............................................................................................................ 6 Strategy: ......................................................................................................................... 6 Folate Receptors: ....................................................................................................... 6-7 Biotin: .......................................................................................................................... 7-8 Materials and Instrumentation: ...................................................................................... 8 Materials: ....................................................................................................................... 8 Instruments: .................................................................................................................. 9 Method and Analysis: ....................................................................................................... 9 Synthesis of Folate Cysteinyl Dithiopyridil 653 (FCD) ............................................. 9 LC Analysis and Purification of FCD .................................................................... 9-10 Lyophilization and LCMS analysis of FCDp .......................................................... 12 Synthesis of PEG-BIOTIN ......................................................................................... 11 Synthesis of Folate-Biotin from FCDp & PEG-BIOTIN....................................... 121 Folate-Biotin Flowchart............................................................................................ 122 Result and Discussion: .................................................................................................... 13 HPLC and LC-MS Analysis of FCD: ........................................................................ 13 Conclusion: ...................................................................................................................... 13 Acknowledgement: .......................................................................................................... 14 References:.................................................................................................................. 15-16 List of Figures: ................................................................................................................ 17 Figure 1: Structure of Folic Acid.. ............................................................................. 17 Figure 2: FR-mediated endocytosis ofa folic acid drug Conjugate.. ...................... 17 Figure 3: Rotatory Evaporator.. ................................................................................ 18 Figure 4: Lyophilizer system buit in GSU by Dr.Henne. ........................................ 18 Figure 5: HPLC (Hewlett Packard Sries 1050). ....................................................... 19 Figure 6: Chromatogram of a typical injection of FCD. ......................................... 20 Figure 7: Collecting FCD peak at 30min. ................................................................. 21 Figure 8: Purified FCD. .............................................................................................. 22 Figure 9: Centrifuge (Thermo Scientific) ................................................................. 23 Figure 10: Basic schematic of FCD from Folate Cysteine and Aldrithiol. ............ 24 Figure 11: Basic schematic to make FCD bound to a drug.. .................................. 24 Figure 12: Vortex mixer used for synthesis and swelling.. ...................................... 25 Figure 13: Total Ion Chromatogram of Liophilized purified FCD.. ...................... 26 4 Abstract: Targeted drug delivery is a newer and less explored arena that has potential scope in tumor targeting because the regular chemotherapy has lethal effects on the normal cells also. Our present Work was an attempt to specifically target receptors on cancerous cells, especially the Folate Receptor (FR). Folate is important for cells for DNA synthesis (1) and the tissues that rapidly divide require folate, cancer cells are no exceptions for this. To cope with the increased demand for the folate the cancer cells express greater FR on the cell surface. This very fact was our basis for the construction of our probe (1,2). Previous work in the group successfully demonstrated the use of dyes like Rhodamine to link folic acid in the construction of the probe. Dyes like Rhodamine have great advantages but are proven carcinogenic(3) so there is need for more safe linking agents like biotin which is a naturally occurring substance in human body. In this study, the concept of disulfide linkage was applied in order to link Folate Cysteinyl Dithiopyridil to Cysteinyl biotin as a Mass Spectoscopic Probe. Because the mechanism in the study directly bonds via disulfide bond it makes linkage possible with various different types of moieties such including DNA. Because FR is overly produced in malignant cells, several types of dyes and small molecules are being studied as to bind with FR. 5 Introduction: Cancer therapy: Cancer till date remains one of the most challenging diseases to the medical society. Many years of Research and huge expenditure of human as well as monetary capital could only achieve so little and hence there is a lot to understand regarding this mind boggling disease. The most baffling problems with this disease are the eclectic nature of the cause of the disease. (4) Strategy: Our aim was to make Folate probe with wider horizons and minimal side effects in this project cystenyle folate was attached to mixed disulfide coupling agents like aldrithirol this could be linked to dyes like rhodamine to increase the detection range visually(5)due to the carcinogenicity of rhodamine(3) it is being replaced by biotin capture probe. Folate receptors: Use of Folate receptors as Trojan horse to deliver micro particles can be dated back to the year 1991 when Dr.Leamon demonstrated the uptake of the folate tagged particles by cells expressing folate.(1) Folate is a must for DNA synthesis, repair, and methylation, also Folate is involved in biological reactions especially that utilize vitamins B6 and B12 as cofactors. Low concentrations of these vitamins can disturb one-carbon metabolism pathways [2] 6 Glycosyl-phosphatidylinositol (GPI) -anchored folate receptors (FR) have a
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