1 FUNCTIONAL NUCLEIC ACID INCORPORATED NANOMATERIALS FOR BIOANALYTICAL AND BIOMEDICAL APPLICATIONS By TAO CHEN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUI REMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2013
2 2013 Tao Chen
3 To m y b eloved p arents, b rother, and h usband
4 AC KNOWLEDGMENTS This dissertation would not have been possible without the help of so many people in so many ways. It is also the product of numerous fortuitous encourters with people who have changed the course of my scientific career. First of all, I would like to gratefully and sincerely thank my research advisor, Dr. We ihong Tan, for his guidance, inspiration, encouragement, and friendship during my graduate study at University of Florida. His mentorship is paramount in shaping me into a critical thinker and an independent scientist. His trust is my spiritual support and confidence source in these difficult times. I am truly fortunate to be a Tan group member and proud to be a gator. I would also like to express my honest appreciation to my committee membe rs, Dr. Ken neth B. Wagener, Dr. Gail E. Fanucci, Dr. Y. Charles Cao, and Dr. Christopher D. Batich for their helpful discussions, invaluable suggestions, and continuous support. I am sincerely grateful to Dr. Daniel Talham, Dr. Nicolo Omenetto, Dr. James C Horvath, and Dr. Z. Hugh Fan for their reference letters. I would also like to show my heartfelt thanks to Dr. James D. Winefordner for the James and Laura Winefordner Graduate Fellowship. I am deeply obliged to Dr. Kathryn R. Williams for her patient re visions to my manuscripts and conference presentations. I would also like to deliver my particular acknowledgement to Dr. Ben Smith for his time and help. All the past and present Tan group members have contributed to my research progress and made my PhD j ourney wonderful. I c annot thank Dr. Mohammed Ibrahim Shukoor, Dr. Quan Yuan and Dr. Suwussa Bamrungsap enough for introducing me to the field of nanomaterials, teaching me experimental skills, and polishing m y research capability during the first two yea rs of my graduate study I sincerely appreciate Dr. Joseph Phillips, Dr. Haipeng Liu, Dr. Ruowen Wang Dr. Kwame Sefah, Dr. Yanrong
5 Wu, Dr. Huaizhi Kang, Dr. Yan Chen, Dr. Ling Meng, Dr. Zhi Judy Zhu, Dr. Xiangling Xiong, and Dr. Mingxu You for their help and training on organic synthesis, nucleic acid chemistry, and molecular biology experiments. I am deeply grateful to Dr. Erqun Song, Dr. Zilong Zhao, Elizabeth Jimenez, Ismail soy, Da Han, Cuichen Sam Wu and Joshua G. Daj ac for their helpful d iscussions and experimental collaborations on my p rojects I am particularly thankful to Dr. Xiaoling Zhang Dr. Yufen Huang, Dr. Liu Yang Dr. Kelong Wang, Dr. Xiaohong Tan Dr. Hongying Jia Dr. Tao Zhang, Dr. Na Shao, Dr. Yan Jin, Dr. Cuisong Z h ou Dr. Hui William Chen, Dr. Youngmi Kim, Dr. Dosung Sohn, Dr. Dalia Lopez Colon, Dr. Hui Wang, Dr. Basri Gulbakan, Dr. Dimitri Van Simaeys, Dr. Jin Huang, Lu Peng, Emir Yasun, Yufei Zhang, Sena Cansiz, Liqin Zhang, Cheng Cui, Carole Champan hac, Huseyin Erdal, Bincheng Yin, Jin g Zheng, Chunmei Li, Bo Liu Liping Qiu, Weijia Hou, Yanyue Wang, I Ting Teng, Benjamin B. Arline, and others for their love and friendship. Finally and most importan t ly I owe a huge debt of gratitude to my family for their support, encouragement, and unwavering love which are the bedrocks upon which the past 26 years of my life have been built. I sincerely appreciate my parents and brother for their unconditional faith and love in me, which gives me the motivation an d confidence to tackle challenges, make progress, and b e successful. I am deeply gratitude to my husband, Weian Sheng, for being a fantastic colleague, friend, and lover. I thank him very much for being always with me, no matter in good times or hard times His great tolerance of my occasional madness is a testament of his unyielding devotion and love to me. Being his wife is the greatest achievement and most wonderful thing of my PhD life.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 L IST OF TABLES ................................ ................................ ................................ .......... 10 LIST OF FIGURES ................................ ................................ ................................ ........ 11 ABSTRACT ................................ ................................ ................................ ................... 14 CHAPTER 1 INTROD UCTION ................................ ................................ ................................ .... 16 Nucleic Acids ................................ ................................ ................................ .......... 16 Composition and Structure of Nucleic Acids ................................ ..................... 16 Chemical Synthesis of Nucleic Acids ................................ ............................... 18 Stability of Nucleic Acids ................................ ................................ .................. 20 Functional Nucleic Acids ................................ ................................ ......................... 22 Aptamers ................................ ................................ ................................ .......... 23 Molecular Beacons ................................ ................................ ........................... 24 Aptamer Molecular Beacons ................................ ................................ ............ 26 Nanomaterials and Their Applications ................................ ................................ .... 27 Magnetic Nanoparticles ................................ ................................ .................... 28 Synthesis of magnetic nanoparticles ................................ .......................... 28 Surface engineering of magnetic nanoparticles ................................ ......... 31 Applications of magnetic nanoparticles ................................ ...................... 32 Graphene Oxides ................................ ................................ ............................. 35 DNA Micelles ................................ ................................ ................................ .... 38 Synthesis of DNA micelles ................................ ................................ ......... 39 Applications of DNA micelles ................................ ................................ ..... 41 2 SMART MULTIFUNCTIONAL NANOSTRUCTURE FOR TARGETED CANCER CHEMOTHERAPY AND MAGNETIC RESONANCE IMAGING ............................. 52 Background ................................ ................................ ................................ ............. 52 Experimental Section ................................ ................................ .............................. 54 Synthesis of Heterobifunctional PEG L igand ................................ .................... 54 PEGylation of Porous Hollow Magnetite Nanoparticles and Doxorubicin loading ................................ ................................ ................................ .......... 55 Synthesis of Aptamer ................................ ................................ ....................... 56 Anchoring of Aptamers onto PEGylated Porous Hollow Magnetite Nanoparticles ................................ ................................ ................................ 56 Doxorubicin Payload Determination and Release Kinetics Study ..................... 57 Binding Test ................................ ................................ ................................ ..... 57 Internalization and Co localization Study ................................ .......................... 58
7 Cytot oxicity Assay ................................ ................................ ............................ 58 Relaxation Measurements and Magnetic Resonance Imaging ......................... 59 Results and Discussion ................................ ................................ ........................... 60 Synthesis and Characterization of Smart Multifunctional Nanostructures ........ 60 Doxorubicin Loading and Release Profile ................................ ......................... 61 Binding and Internalization behavior of Smart Multifunctional Nanostructures ................................ ................................ .............................. 62 Cell Viability and Proliferation Assay ................................ ................................ 64 Explore the Potential of Smart Multifunctional Nanostructures for Magnetic Resonance Imaging ................................ ................................ ...................... 66 Conclusions ................................ ................................ ................................ ............ 67 3 SEMIQUANTIFICATION OF ATP IN LIVE CELLS USING NONSPECIFIC DESORPTION OF DNA FROM GRAPHENE OXIDE AS THE INTERNAL REFERENCE ................................ ................................ ................................ .......... 76 Background ................................ ................................ ................................ ............. 76 Experimental Section ................................ ................................ .............................. 77 DNAs ................................ ................................ ................................ ................ 77 Preparation of Graphene Oxide ................................ ................................ ........ 77 Fluorescence Response of ATP Aptamer Molecular Beacon and Control Molecular Beacon to ATP in Buffer ................................ ............................... 78 Live Cell Imaging of ATP Using Graphene Oxide to Deliver DNAs into Cells .. 78 In Situ Semiquantification of ATP ................................ ................................ ..... 79 Results and Disscussion ................................ ................................ ......................... 79 Nonspecific Desorption of ssDNA from DNA/Graphene Oxide Complex by Proteins ................................ ................................ ................................ ......... 79 Test the Feasibility of the Design in Buffer ................................ ....................... 81 Intracellular ATP Detection Using ATP Aptamer Molecular Beacon/Graphene Oxide Complex ................................ ................................ 82 Intracellular ATP Semiquantification through Raitometric Measurements ........ 83 Conclusions ................................ ................................ ................................ ............ 85 4 DNA MICELLE FLARES FOR INTRACELLULAR MRNA IMAGING AND GENE THERAPY ................................ ................................ ................................ ............... 94 Back ground ................................ ................................ ................................ ............. 94 Experimental Section ................................ ................................ .............................. 96 Synthesis of Lipid Phosphoramidite ................................ ................................ 96 Synthesis of Oligonucleotide Probes ................................ ................................ 97 Critical Micelle Concentration ................................ ................................ ........... 98 Agarose Gel Electrophoresis ................................ ................................ ............ 98 Dynamic Light Scattering and Zeta potential Measurements ........................... 99 Fluorescence Measurements ................................ ................................ ........... 99 Cell Culture ................................ ................................ ................................ ....... 99 Cell Lysate Preparation ................................ ................................ .................... 99 Confocal Laser Scanning Microscopy Experiments ................................ ....... 100
8 Flow Cytometry Experiments ................................ ................................ .......... 100 Cytotoxicity Assay ................................ ................................ .......................... 101 Results and Discussion ................................ ................................ ......................... 101 Synthesis and Characterization of Molecular Beacon Micelle Flares ............. 101 Evaluate the Performance of Molecular Beacon Micelle Flares in Buf fer System ................................ ................................ ................................ ........ 102 Evaluate the Performance of Molecular Beacon Micelle Flares in Living Cells ................................ ................................ ................................ ............ 103 Molecular Beacon Micelle Flares f or Gene Regulation ................................ ... 105 Conclusions ................................ ................................ ................................ .......... 106 5 ONE STEP FACILE SURFACE ENGINEERING OF HYDROPHOBIC NANOCRYSTALS WITH DESIGNER MOLECULAR RECOGNITION ................. 116 Background ................................ ................................ ................................ ........... 116 Experimental Section ................................ ................................ ............................ 117 Synthes is and Characterization of Hydrophobic Nanocrystals ....................... 117 Synthesis of hydrophobic nanocrystals ................................ .................... 117 Transmission electron microsco py images of hydrophobic nanocrystals 122 Synthesis and Characterization of Amphiphilic Oligonucleotides ................... 122 Synthesis and Chara cterization of Functionalized Hydrophobic Nanocrystals ................................ ................................ ............................... 123 Synthesis of functionalized nanocrystals ................................ ................. 123 Characterization of func tionalized nanocrystals ................................ ....... 123 Binding of Functionalized Hydrophobic Nanocrystals with Nucleic Acid Targets ................................ ................................ ................................ ........ 125 Binding of Function alized Hydrophobic Nanocrystals with Cancer Cell Target ................................ ................................ ................................ .......... 126 Magnetic Resonance Imaging ................................ ................................ ........ 126 Results and Discussion ................................ ................................ ......................... 127 Development and Characterization of the Surface Engineering Approach ..... 127 Tunable Molecular Recongition of Engineered nanocrystals .......................... 129 Explore the Potential of Modified Nanocrystals for Magnetic Resonance Imaging ................................ ................................ ................................ ....... 131 Comparison Between Current Method and Traditional Polymer Systems ...... 132 Conclusions ................................ ................................ ................................ .......... 132 6 SUMMARY AND FUTURE DIRECTIONS ................................ ............................ 142 Functional Nucleic Acid Incorporated Nanomaterials for Bioanalytical and Biomedical Applications ................................ ................................ ..................... 142 Smart Multifunctional Nanostructure for Targeted Cancer Chemotherapy and Magnetic R esonance Imaging ................................ .............................. 143 Semiquantification of ATP in Live Cells Using Nonspecific Desorption of DNA from Graphene Oxide as the Internal Reference ................................ 143 DNA Micelle Flares for Intracellular mRNA Imaging and Gene Therapy ........ 144
9 One Step Facile Surface Engineering of Hydrophobic Nanocrystals with Designer Molecular Recognition ................................ ................................ 145 Future Directions ................................ ................................ ................................ .. 146 In vivo Applications of Smart Multifunctional Nanostructure and DNA/Graphene Oxide System ................................ ................................ .... 146 All in One DNA Micelles for Cancer Targeting, Imaging, and Therapy .......... 146 LIST OF REFERENCES ................................ ................................ ............................. 148 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 168
10 LIST OF TABLES Table page 2 1 Detailed aptamer sequence information. ................................ ............................ 69 4 1 Detailed sequence information for all oligonucleotide probes. .......................... 107 5 1 Detailed sequence information and CPG selection for amphiphilic DNA. ......... 134 5 2 DLS and zeta potential measurements of Fe Fe 3 O 4 CSNPs engineered with chimeric DNA molecules of different lengths and sequence information. ......... 134
11 LIST OF FIGURES Figure page 1 1 The composition and structure of nucleic acids. .. ................................ ............... 44 1 2 Chemica l synthesis of nucleic acids. ................................ ................................ 45 1 3 Representative base modifications for nucleic acids. 14 ................................ ....... 46 1 4 Schematic illustration of cell b ased SELEX. ................................ ....................... 46 1 5 Working p rinciple of MBs and AMBs. ................................ ................................ 47 1 6 Schematic illustration of typical m ethods for MNP synthesi s. ............................ 47 1 7 Surface engineeri ng of MNPs. ................................ ................................ ........... 48 1 8 Applica tions of MNPs. ................................ ................................ ........................ 49 1 9 Applications of GOs. ................................ ................................ ........................... 50 1 10 Applications of DNA micelles. ................................ ................................ ............. 51 2 1 Synthesis route for heterobifunctional PEG ligand. ................................ ............ 70 2 2 Synthesis and characterization of SMNs ................................ ........................... 71 2 3 Mechanism of SMNs for targ eted cancer chemotherapy. ................................ ... 72 2 4 Cumulative release of DOX from DOX SMNs at different pH values. . ............... 72 2 5 Flow cytometry histograms to monitor the binding of SMNs to CEM cells (target cells) and Ramos cells (control cells) . ................................ .................... 73 2 6 Confocal fluorescence microscopy images to monitor the binding of SMNs to CEM cells and Ramos cells . ................................ ................................ .............. 73 2 7 Co loca liz ation study of sgc8 aptamer and SMNs with lysose nsor in CEM cells ................................ ................................ ................................ ................... 74 2 8 Cytotoxicity as say of CEM cells and Ramos cells treated with SMN only, DOX only, and DOX SMNs. ................................ ................................ ................ 74 2 9 Potential of using SMNs as T 2 contrast agents. .. ................................ ............... 75 3 1 Nonspecific d esorption of ssDNA from GO by n ontarget p roteins. .................... 86 3 2 TEM image of GO. ................................ ................................ .............................. 86
12 3 3 Fluorescence spectra of FAM labeled ssDNA library o nly and FAM labeled ssDNA library/GO complex in the presence of di ffe rent concentrations of BSA. ................................ ................................ ................................ .................. 87 3 4 Fluorescence intensity of FAM labeled ssDNA library/GO complex in the presence of different concentrations of FBS. ................................ ..................... 87 3 5 Confocal microscopy of HeLa cells treated with ATP aptamer/GO under two different conditions . ................................ ................................ .......................... 88 3 6 Fluorescence spectra of AAMB a nd CMB in the presence of AT P. ................... 88 3 7 Comparison of fluorescence intensities of AAMB and ATP aptamer under d ifferent conditions. ................................ ................................ ........................... 89 3 8 Intracellular imag in g of ATP. .. ................................ ................................ ............. 89 3 9 Confocal fluorescence microscopy of HeLa cells treated with AAMB/GO at different concentrations of GO. . ................................ ................................ ......... 90 3 10 Confocal fluorescence microscopy of HeLa cells treated with AAMB/GO for 3 h and after further different incubation tim es. ................................ .................... 91 3 11 Advanced ATP i m aging p robe d esign. ................................ .............................. 92 3 12 Confocal microscopy of HeLa ce lls treated with advanced ATP imaging probe. . ................................ ................................ ................................ ............... 92 3 13 ATP imaging with drug stimulation. ................................ ................................ ... 93 3 14 Intracellular imaging quantification . ................................ ................................ ... 93 4 1 Schemati c illustration of MBMFs for intracellular mRNA det ection and gene therapy. ................................ ................................ ................................ ........... 108 4 2 Detailed synthesis route for lipid phosphoramidite. ................................ .......... 108 4 3 Characterization of MBMFs. ................................ ................................ ............ 109 4 4 Purification and characterization of L MBs which can self assemble into MBMFs. ................................ ................................ ................................ ........... 109 4 5 CMC de termination of MBMFs. ................................ ................................ ....... 110 4 6 Perfo rmance evaluation of MBMFs in buffer system. ................................ ....... 111 4 7 Condition optimization of MBMFs in living cells. . ................................ ............. 112 4 8 Co localization assay of MBMFs w ith Tf Alexa 633 ................................ ......... 112
13 4 9 Investigation of MBMFs in living cells. ................................ ............................. 113 4 10 Pe rformance comparison between MBMFs and S MBMFs in buffer system. .. 114 4 11 CMC determination of S MBMF s. ................................ ................................ .... 114 4 12 Cytotoxicity assay of A549 cells treated with S MBMFs and non complementary S MBMFs. ................................ ................................ ............... 115 5 1 Strategy for one step surface engineering of hydrophobic nanocrystals with designer molecular recognitio n. ................................ ................................ ....... 135 5 2 Characterization of chimeric DNA molecule engineered Fe Fe 3 O 4 CSNPs . .... 136 5 3 DLS measurements of modified Fe Fe 3 O 4 CSNPs in water (red) and unmodified Fe Fe 3 O 4 CSNPs in hexane (black). ................................ ............. 136 5 4 UV Vis spectra of modified Fe Fe 3 O 4 CSNPs in water (red) and unmodified Fe Fe 3 O 4 CSNPs in hexane (black). . ................................ ............................... 137 5 5 FT IR spectra of modified (red) and unmodified (black) Fe Fe 3 O 4 CSNPs. ..... 137 5 6 Fluorescence intensity and the number of ligand per e ngineered nanoparticle as a function of chimeric DNA molecule concentration for Fe Fe 3 O 4 CSNPs modified with lipid T20. ................................ ................................ ..................... 138 5 7 Fe Fe 3 O 4 CSNPs treated with different co ncentrations of lipid T2 0 ................ 138 5 8 Fe Fe 3 O 4 CSNPs treated with chimeric DNA molecules of different lengths and sequence information. ................................ ................................ ............... 139 5 9 Surface eng ineering of various hydroph obic nanocrystals with different size, composition, and morphology with chimeric DNA molecule mo dification. ....... 139 5 10 Check the presence of empty micelles. . ................................ .......................... 140 5 11 Hybridization between chimeric DNA molecule engineered Fe Fe 3 O 4 CSNPs with their cDNA. . ................................ ................................ .............................. 140 5 12 Specific binding of chimeric DNA molecule engineered Fe Fe 3 O 4 CSNPs to target cancer c ells. ................................ ................................ .......................... 141
14 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements f or the Degree of Doctor of Philosophy FUNCTIONAL NUCLEIC ACID INCORPORATED NANOMATERIALS FOR BIOANALYTICAL AND BIOMEDICAL APPLICATIONS By Tao Chen August 2013 Chair: Weihong Tan Major: Chemistry Advances in nanotechnology and biotechnolo gy are creatin g novel tools for various bioanalytical and biomedical applications. Through rational engineering nanomaterials can be tailored in a predictable manner to meet the needs of specif ic applications. In addition, by coupling with biomolecules (e.g., nucleic a cids) nano materials can possess molecular recognition capability. T herefore, the objective of this research is to develop functional nucleic acid incorporated nanomaterials for early disease diagnosis, targeted drug delivery, and effective cancer therapy. First, we constructed a smart multifunctional na nostructure (SMN) from a porous hollow magnetite nanoparticle (PHMNP), a heterobifunctio nal PEG ligand, and an aptamer for targeted cancer chemotherapy and magnetic resonance imaging (MRI) The PHMNPs were l oaded with the anticancer drug doxorubicin. Using an aptamer with specific binding and efficient internalization to target cells, increased therapeutic efficiency was observed. In addition the SMNs showed great potential for use in MRI as T 2 contrast agen ts Second, a DNA/graphene oxide system compose d of an internal reference probe, an ATP aptamer molecular beacon probe, and graphene oxide (GO) was fabricated for
15 intracellula r ATP monitoring. GO was used to deliver the nucleic acid probes into living cell s an d semiquantitation of ATP in HeL a cells was achieved through ratiometric measurements. Third, DNA micelles self assembled from amphiphilic oligonucleotides containing a hydrophobic diacyllipid and a hydr ophilic oligonucleotide were developed. These DN A micelles po ssess two intriguing properties : ability to en ter living cells and resistan ce to enzymatic digestion, as a result of their hig hly packed DNA nanostructures. M olecular beacon micelle flares (MBMFs) from diacyll ipid molecular beacon conjugate se lf assembly were prepared and utilized for combined messager RNA detection and gene therapy. L ike pyrotechnic flares that produce brilliant light when activated, MBMFs undergo a significant burst of fluorescence enhancement upon target bi nding. This hybrid ization subsequently induces gene silencing, leading to cancer cell apoptosis. Finally, inspired by the first and third project s we developed a one step facile method for hydrophobic na noparticle surface engineering using amphiphilic oligonucleot ides. The procedure is simple and versatile, generating individual nanoparticles with multiple liga nds that can induce multivalent effect. More importantly, by chan g ing the sequence information of oligonucleotides, modified nanoparticles can actively targe t various molecular addresses, ranging from nucleic acids to membrane proteins on cancer cells.
16 CHAPTER 1 INTRODUCTION Nucleic Acids Ev ery organism on e arth contains the molecular instructions for life called nucleic acids. They are complex molecules th at consist of many components, a portion of which are passed from parent organisms to their offspring during the process of reproduction a rose, and the way in whi ch bacteria infect a lun g cell Thanks to the continuous effort of numerous researchers the structure of nucleic acids was elucidated by Watson and Crick in 1953 1 -almost 100 years since the discovery of nucleic acids by Miescher in 1869. From then on, researchers have made many great strides in understa nding n ucleic acids, resulting in applicati ons in a broad spectrum of fields, including biology, medicine, and nanotechnology. 2 4 Here in this chapter, the composition, st ructure, chemical synthesis, and stability of nucleic acid s will be reviewed. Composition and Structure of Nucleic Acids At the most basic level, nucleic acids are composed of a series of smaller molecules called nucleotides (Figure 1 1 A ) Depending on the nucleotides involved, nucleic acids can be largely divided into two types: ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Each nucleotide has thre e components: a phosphate group, either a ribose sugar in RNA or a deoxyr ibose sugar in DNA, and a n itrogen containing base. 5 There are two basic kinds and five sub typ es of nitrogenous bases: the purines with two fused rings, including adenine (A) and guanine (G), as well as pyrimidines with a single ring, including cytosine (C), thymine (T), and uracil (U). RNA contains only A, G, C and U, whereas DNA contains only A, G, C, and T.
17 Even though nucleotides derive their names from the nitrogenous bases they contain, they owe much of their structure and bonding capabilities to their 5 membered sugar ring s. These rings contain 4 carbons and 1 oxygen as shown in Figure 1 1 A the up is attached Accordingly, the the area surrounding the other side of the sugar ring is denote d as the ucleic one through phosphodiester bond and this alternating sugar phosphate arrangement forms the backbone of nucleic acids (Figure 1 1 B ) Nucleic acids are often found as single stranded polynucleotides. However, they assume their most stable form s when double stranded via hydrogen bonds betwe en complementary nitrogenous bases Rather than random base to base bonding, each A pairs only with U (in RNA) or T (in DNA) and each G always pairs with C. 6 There are 2 hydrogen bonds between A and U (in R NA) or T (in DNA), whereas 3 hydrogen bonds are observed between G and C. It is this mutual recognition of A by U (in RNA) or T (in DNA) and G by C that maintains the fidelity of biological heredity and the foundation of genetic s research. Resulting from this pattern of bonding, the double stranded nucleic acids look much like ladders with sugar phosphate side suppo rts and base pair rungs (Figure 1 1 B ) It is worth noting that the sugar phosphate ends of the two polynucleotides that make up double stranded nucleic acid s are arranged in opposite orientations, which mean s that one st r phosphate chain runs in like
18 structure described above, X ray diffraction images taken by Rosalind Franklin in 1952 revealed that nucleotides in double stranded nucleic acids were arran ged in a spiral helix structure (Figure 1 1 B ) 7 Chemical Synthesis of Nucleic A cids Based on the understanding of phosphodiester linkage formation in nucleic acid s and inspired by the automated chemical synthesis of peptides in solid phase, the Khorana group at the Institute for Enzyme Research in U niversity of Wisconsin explore d the chemical synthesis of both DNA and RNA. T hey successfully synthesized DNA and RNA using polystyrene beads as solid support, protected nucleotides as monomers, and N dicyclohexylcarbodiimide ( DCC ) as condensing reagent. 8 In this method, the synthe sis of a DNA or RNA sequence proceeds in the same direction as in nature: from largely limited and more significantly, this method was tedious and inefficien t. This synth etic method was lasted for more than 10 year s until a bre akthrough was achieved in 1983 by the Caruthers group, who designed the synthesis of long nucleic acid sequenc es with high efficiency 9 The novel synthetic approach was based on the use of controlled pore glass (CPG) beads as the solid support, protected phosphoramidite s as monomers, and tetrazole as catalytic reagent. Similar to pro phosphoramidite s are normal nucleotid e s with protection groups for their active amine, hydroxyl, and phosphate groups. These protection groups can prevent undesired side reactions and are removed after the synth esis. Unlike the Khorana method the link to the solid support in the Caruthers process and the synthesis proceeds
19 In Caruthers method, a typical cycle of the chemical synthesis of nucleic acids, taking a DNA sequence as an e xample, includes four steps: de protection, coupling, capping, and stabilization (Figure 1 2) 9 In the de protection step, the trityl group trichloroacetic acid (TCA), leaving the reactive hydroxyl group for adding the next base. In the coupling step, tetrazole, a weak acid attacks the coupling phosph oramidite nucleoside, forming a tetrazolyl phosphoramidite intermediate which then reacts with the active hydr oxyl group obtained from the de protection group. 10 This coupling step results the to DCC, the use of TCA increases the coupling efficiency to greater than 99%, making the Caruthers method feasible for synthesizing relative long nucleic acid sequences. However, since the coupling efficiency i s not 100%, a coupling failure results in an oligonucleotide would lead to a missing base in the synthesis. Therefore, in the capping step, coupling failures are rem oved before adding the next base by reacting with an acetylating reagent composed of acetic anhydride and N methylimidazole. The last step in the synthesis is oxidation which stabilizes the phosphate linkage. In order to make phosphate linkage stable, iodi ne and water are added to make the phosphate group pentavalen t and form the phosphodiester bond. These four steps are repeated many times until the desired DNA sequence is obtained. At the end of the synthesis, the DNA sequence attach ed to the l group. In addition, protecting groups remain on the four bases to maintain the integrity of ring structures. The pr otecting groups of these
20 four bases are then removed by base hydrolysis using ammonium h ydroxide at high If special bases are used, the de protection procedure varies with the structures and properties of the new bases employed. The de protected DNA sequences are normally purified using desalting, HPLC (High Pressure Liquid Chromatography ), and PAGE (PolyAcylamide Gel Electrophoresis), depending on the lengths and expected applications The purified DNA sequences are then quanti fied by absorbance measurements at 260 nm. Stability of Nucleic Acids Due to the fixed base pairing between A and T (in DNA) or U (in RNA), C and G, a single stranded nucleic acid sequence can specifically hybridize to its complementary target. This is one of the most selective molecular recognition processes in nature as some of the sequences even possess the abili ty to differentiate single base mismatch. Inspired by this specific molecular recognition with high affinity and e nabled by the high efficiency synthesis of oligonucleot ides numerous nucleic acid probes have been synthesized for a broad spectrum of bioana lytical and biomedical applications. For example, an nucleic acid sequence complementary to a part of an DNA or a RNA of the genome can selectively interfere with the process dependent on that segment through otein expression level, inhibited cancer cell growth, etc. 11 12 Many of t hese applications need to be carried out in complex media, such as in living cells, requiring that the nucleic acid probes be stable in the cellular environment Unfortunately, nucleic acid probes can be quickly degraded by a wide variety of n ucleases. In fact, the half lives of most RNA probes in serum are minutes or less. DNA probes are relatively more stable, with half lives up to hours. 13 To increase the stability
21 of nucleic acid probes, chemical modifications are introduced into nucleotides to interfere with the enzymatic mechanisms of nuclease s C hemical modifications for nucleotides ca n involve the phosphodiester li nkage, the base, or the sugar. 14 Currently ther e are 3 kinds of phosphodiester linkage modification: phosphorothioate modification (the exchange of one non bridging oxygen atom with a sulfur atom), 15 phosphorodithioate modification (the exchange of both non bridging oxygen atom s with sulfur atoms), 16 and methylphosphonate modification (the exchange of one non bridging oxygen atom with a methyl group). 17 Among them, the phosphorot hioate and phosphorodithioate m o dication s signi ficantly increase the stability of nucleic acid probes towards several exonu cleases and restriction enzymes, both the phosphorothioate and methylphosphonate modification s change the ch irality of nucleic acid probes, and methylphosphonate modification makes nucleic acid probes non ionic. The heterocyclic ring of purine and pyrimidine bases provides hydrogen bonding functional groups in nucleic acids. Therefore, careful designs are needed for introducing modifications to bases. To date, scientists have succe ssfully synthesized a series of modified bases, with representative structures shown in Figure 1 3 14 When properly designed, the incorporation of modified bases into nucleic acid sequences can attenuate or even elim inate the molecular recognition between nucleases and the modified nucleic acid sequences, leading to their increased enzymatic resistance. On the other hand, the use of modified bases can provide useful information on the importance of specific functional groups in natural bases. For example, 7 deazapurine nucleosides, which lack the N 7 nitrogen atom, are valuable probes for studying the role of N 7 nitrogen in a wide range of molecular recogniti on processes including the
22 interaction between trp repressor and trp EDCBA operon, and the binding between restriction enzymes and nucleic acids. 18 19 Sugar modified nucleic acids possess sup erior stability toward nuclease digestion, making them better candidates than natural nucleic acids for a series o f biomedical applications. In addition, they also have been used extensively to study hydroxyl groups in catalysis. 14 20 Compared to natural nucleic acids, many of the aforementioned modificat ions have resulted in nucleic acids with much greater stability and s ignificantly elongated half lives in complex med ia. 12 This advancement together with the rapid development of oligonucleotide synthesis have greatly expand ed the pool of nucleic acids and improved the ir prope rties for desired bioanalytical and biomedical applications. Functional Nucleic Acids Inspired by Darwinian evolution variatio n, selection, and reproduction the Szostak group 21 as well as the Gold group 22 found that it was possible to select RNA sequences capable of recognizing a given small molecule or protein from a random RNA sequence pool. Since then there has been an explosion of research devoted to the development of increasing ly sophisticated and useful nucleic acids, collectively referred to as functional nucleic acids. Members of the functional nucleic acid family include : aptamers, molecular beacons, aptamer molecular beacons, DNAzymes, and ribozymes. 23 The applications of these functional nucleic acids have covered diverse topics such as biosensor development, targeted drug deli very, and effective cancer therapy. In this section, a brief review of aptamers, molecular beacons, and aptamer molecular beacons will be given.
23 Aptamers which means fit and the Greek which means region, are short s equences of oligonucleotides or pepti des that specifically bind to their targets. In this dissertation, the main focus is nuc leic acid aptamers generated from a process known as SELEX (Systematic Evolution of Ligands by EXponential enrichment). 21 22 By folding into distinct secondary or tertiary structures, aptamers can specific ally bind to their ta rgets, vary in g from small molecules 24 25 proteins 26 28 to cells 29 and tissues 30 with high affinity. Compared to antibodies, t he t involve animals. Theref ore, the targets of aptamers can li terally be anything, even toxic molecules. Recently, the Tan lab has developed a technique called cell based SELEX that can generate a panel of apta mers for living cells (Figure 1 4) 29 For preparati on, a library with 10 13 10 16 single stranded oligonucleotides is constructed. These oligonucleotides have the same length and are designed to have a random sequence (30 40 bases) flanked by two primer regions (18 20 bases). In a typ ical cycle for cell ba sed SELEX the library is first incubated with target cells at 4 C, the temperature that can effectively inhibit undesired oligonucleotide internalization. During this step, some sequences bind to the targ et cells with high affinity, while others interact weakly or not at all with their target cells Therefore, in the second step, the bound sequences and the unbound sequences are physically separated and only the ones bind ing to the target cells are collected. To ensure that they bind to membrane componen ts specific to the target cells, in the third step, the eluted sequences are incubated with control cells at 4 n the fourth step, the bound sequences and the unbound sequences are also physically separated but only those that
24 of the entire selection largely depends on the second and fourth step s, since narrowing the pool with high affinit y sequences for target cells will greatly speed th e enrichment process. After the fouth step the eluted ssDNA sequences are considered as the enriched pool and used as the library for the next round after polymerase ch ain reaction ( PCR ) amplification. This cycle is typically repeated 15 to 25 times until the pool is enriched to the desired level. Then the enriched pool is cloned and sequenced to identify a panel of potential aptamers. At last, these aptamer candidates a re chemically synthesized, appropriately labeled, and systematically tested to gen erate the best a ptamers for target cells with minimal interaction with control cells. Using this process, the Tan lab has generated many specific aptamers for several types of diseased, infected, and cancerous cells, including lymphocytic leukemia 31 myeloid leukemia 32 liver cancer 33 lung cancer 34 35 colorect al cancer 36 and others 37 38 The merit of cell based SELEX lies in the generation of cell specific molecular probes without knowledge of cell membrane signatures And the advantages of aptamers for bioanalytical and biomedical applications lie in their easy preparation, facile modification, good stabi lity, efficient penetration and minimal immune response. 39 Molecular Beacons Molecular beacons (MBs) first described in 1996 by Sanjay Tyagi and Fred Russel Kramer 40 are oligonucleotide sequences composed of a target recognition region (about 15 30 bases) flanked by two short complementary stem sequences (about 5 8 base pairs) (Figure 1 5 A ) 41 These sequences are dual labeled with a fluorophore on one end and a quencher on the othe r end. In the absence of target MB s are forced to form a hairpin (stem loop) structure due to the base pairing of st em sequences This brings the fluorophore and quencher into close proximity, thereby quenching
25 fluorescence effectively. I n the presence of targets, the stronger intermolecular hybridization between the target and the loop overcomes the weaker stem base pa iring, physically and spatially separating the fluorophore and the quencher to restore fluorescence. Through rational sequence design and careful fluorophore/quencher selection, the performances of MB s can be easily tuned for desired applications. The sup erior flexibility of design, together with the suitable thermodynamic stability and the efficient signal switching has greatly facilitated the use of MB s in disease diagnosis, 42 living system analysis 43 enzymatic process i nvestigation, 44 and effective cancer therapy. 45 Among all these applications of MB s, the use for living system analysis is of particular interest. Because of their distinctive signaling mechanism, MB s ar e excellent candidate s for intracellular monitoring for several reasons : 46 (1) Targets are detected in real time without the need to separate bound and unbound probes. This is of great significance for intrac ellular studies sin ce it is impossible to remove the excess unbound probes without destroying living cells. (2) MB s are highly sensitive, making them capable of sensing trace amount s of biomolecules in living cells, such as mRNA with a low copy number. (3 ) MB s have exceptional selectivity that can differentiate between perfectly comple mentary targets and mismatched targets. However, several important issues should be addressed when designing MB s for living cell analysis: (1) MB s should be designed to find their targets in living cells. For example, mRNA sequences are normally very long and have complicated secondary structures. Therefore, the target sites should be chosen in the regions that have a high probability of remaining single stranded structures t o ensure that MB binding is
26 thermodynamically favorable. 46 (2) An effective method is needed for efficient delivery of MB s into cells. Currently, the available methods include microinjection, 47 electroporation, 48 reversible permeabilization 49 and transfection reagen t assisted delivery 50 (3) MB s are required to have desired stability, including resistance to cellular nucleases and immune to nonspecific interactions. Since natural nucleot ides are prone to enzymatic digestion, chemically modified nucleotides are employed to synthesize molecular beacons with enhanced stability 51 52 In addition, various nanomaterials have been used to protect MB s before reaching t heir intracellular targets 53 54 Aptamer Molecular Beacons Even though MBs were originally developed for nucleic acid analysis, they have recen tly shown promise in monitoring o ther biomolecules, such as ATP, thrombin platelet derived growth factor ( PDGF ) and others 41 55 Generally, this is achieved by incorporating aptamer sequences into the design of m olecular beacons (Figure 1 5 B ) Through this, the built in signal transduction mechanism of molecular beacons, excellent selectivity of aptamers to a broad spectrum of targets. This greatly simplified the sensing strategies for nongenetic targets, especially proteins. For example, detection of proteins in vitro through dye labeled antibodies involves complicated separation procedures (e.g., gel electrophoresis) and monitoring proteins in vivo through green fluorescence protein (GFP) fusion requires gene clone techniques w hich are not always successful 56 In addition, aptamer molecular beacons (AMBs) can not only report the presence but also quantify the amount of specific proteins. To directly transduce molecular recognition event s into optical signals, AMB s are designed by adding additional sequences to aptamers so that they can form a stable
27 hairpin (stem loop) structure in the absence of target The ideal AMB s have the lowest fluorescence background without targets but the highest fluorescence signal with targets. Therefore rational design is needed for constructing AMB s based on the sequence information and secondary structure of aptamers. Nanomaterials and Their Applications Nanomaterials are materials having a size of 100 nm or sma ller in at least one dimension Because of quantum effects stemm ing from the large surface area to volume ratio, nanomateri als possess unique optical, electronic, magnetic, mechanical, physical, and chemical properties. 57 With these unique prope rties, nanomaterials have shown great potential for numerous applications, rang ing from energy harvesti ng to information technology and biomedical research 57 With recent pro gress on nanomaterial synthesis and surface engineering, various nanoparticles have been increasingly us ed in biosensor development, molecular imaging, drug delivery, and cancer therapy 58 61 These include noble metallic nanoparticles (e.g., gold nanoparticles, silver nanoparticles, iron oxide nanoparticles), semiconductor quantum dots, silica based nanoparticles (e.g., dye doped silica nanoparticles, mesoporous silica nanoparticles), carbon based nanoparticles (e.g., carbon nanotubes, graphene oxides), polymeric nanoparticles, liposomes and micelles. Through surface modification, the biocompatibility of nanoparticles can be greatly improved, especially those sy nthesized in organic solvents. In addition, by functionalizing nanoparticles with specific biomolecules, the resulting conjugates will also possess the properties of these biomolecules. For example, the conjugation of aptamers onto nanoparticles will provi de the resulting conjugates with selective targeting capability to various cancer cells. 57 Interestingly, compared to free aptamers,
28 aptamer conj ugated nanoparticles have even higher binding af finity to target cancer cells. 62 Similarly, through the incorporation of MBs or AMBs the modified nanoparticles will have signal rea douts for different kinds of genetic or nongenetic targets. 53 This section includes a brief review of magnetic nanoparticles, graphene oxides, and DNA micelles, including their chemical synthesis, surface modification, a nd biological applications. Magnetic Nanoparticles Magnetic nanoparticles (MNPs) are made from magnetic materials, such as Co, Ni, Fe, Fe 2 O 3 Fe 3 O 4 and others. 63 Due to their unique properties, MNP s are of great interest for researchers from a broa d spectrum of areas, including heterogeneous catalysis, environment science, data storage, and biomedical engineering. 64 Even though va rious methods have been reported for the synthesis of MNP s with different compositions, their successful application in the fields listed above is largely dependent on the magnetization value, the size and size distribution, as well as the surface chemistr y of MNP s. For example, MNP s in the size range of 6 15 nm a re rapidly removed through extr avasations and renal clearance. 65 In addition, MNP s with narrow size distribution h ave uniform pharmac okinetics and bio distribution. In the following, after discussing the synthesis of MNPs we will focus on recent development in the surface modifi cation of MNP s as well as their biological applications. Synthesis of magnetic nanopartic les MNP s with a number of different compositions, including Fe, Co, Fe 2 O 3 Fe 3 O 4 MgFe 2 O 4 MnFe 2 O 4 CoPt 3 a nd FePt, have been synthesized. 66 In the past decades, much research has been devoted to the efficient synthesis of shape control led and
29 mono disperse MNP s. Typical methods are co precipitation, t hermal decomposition, and micro emulsion. Co precipitation is a simple approach to syn thesize MNPs mainly Fe 3 O 4 Fe 2 O 3 MNPs from aqueous Fe 2+ /Fe 3+ salt solut ions by the addition of a base (Figure 1 6 A ) 65 Generally, Fe 3 O 4 nanoparticles are prepared by adding a base to a mixture of FeCl 2 and FeCl 3 with a molar rati o of 1:2. In order to achieve complete precipitation of Fe 3 O 4 nanoparticles, the pH of the solution should be maintained between 9 and 14 by the added bas e. In addition, a n oxygen free environment is required to control the reaction kinetics, prevent possible oxidation, and avoid particle agglomeration. The size, shape, and composition of MNP s obtained by the co precipitation method are greatly affected by the salt type (e.g., chlorides, sulphates, nitrates, etc.), the Fe 2+ /Fe 3+ ratio the reaction temperature, the pH value, and the ionic strength of the solution. 65 Even though the co precipitation method is fully reproducible, the experi m ental challenge lies in the control of particle size and shape. MNP s prep ared by co precipitation unfortunately tend to be rather poly disperse and irregular. To this end, researchers are using organic additives (e.g., polyvinyl alcohol (PVA)) as st abiliza tion/reducing agents. 67 Inspired by the synthesis of high quality semiconductor nanocrystals, thermal decomposition can be used to synthesize various MNP s, includin g metal oxid e nanoparticles, 68 metallic nanoparticles, 69 and m etal alloy nanoparticles, 70 with excellent control over size and shape. Generally, thermal decomposition is realized by heating organometallic precursors in organic solvents containing stabilizing surfactants at high temperatures (Figure 1 6 B ) These organometallic precursors include metal
30 acetylacetonates (M(acac) n M = Fe, Mn, Co, Ni, Cr; n=2 or 3, acac = acetylacetonate), metal cupferronates (M x Cup x M = metal ion ; cup = N nitrosophenylhydroxylamine, C 6 H 5 N(NO)O ), and metal carbonyls. 66 Typical surfactants can be fatty acids, ole ic acid, and hexadecylamine. 66 The ratio of the starting reagents, including organometallic precurso rs, surfactants, and solvents play s a major role in controlling the size and shape of MNP s. 66 T he reaction time, aging period, and reaction temperature can also influence the size and shape of MNP s 66 When synthesizin g metal oxide nanoparticles, if the metal in the organometallic precursor is zerovalent, such as in metal carbonyls, thermal decomposition first leads to the formation of metallic nanoparticles, then produces metal oxide nanoparticles through the addition of oxidant, for example, (CH 3 ) 3 NO; 71 if the organometallic precursor contains a cationic metal center, thermal decomposition directly gener ate s metal oxide nanoparticles. 72 Currently, monodisperse metal oxide nanoparticles, with sizes adjustable over a wide range 3 to 50 nm can be synthesized through thermal decomposition, with controll ed shapes, such as dots, cubes, and rods. 73 In comparison, met a llic nanoparticles prepared through thermal decomposition are only available in the size range of 2 to 10 nm. 74 In addition, some of these metallic nanoparticles, for in stance, iron nanoparticles, can be easily oxidized by exposu re to air. 74 Metal alloy nanoparticles have many advantages, including high magnetic anisotropy, enhanced magn etic susceptibility, and large coercivities. Through thermal decomposition, various metal alloy nanoparticles, such as CoPt 3 75 FePt, 70 FeP, 76 MnP 77 nanoparticles, can be synthesized.
31 A microemulsion, is a thermodynamically stable isotropic dispersion of two immiscible liquids, where the microdomain s of eithe r or both liquids are stabilized by an interfacial film of surfactant molecules. 78 Due to the presence of microdomains, microemulsions can be used as reactors to synthesize na noparticles. So far, Co, CoPt 3 and Au coated CoPt 3 nanoparticles have been prepared usin g water in oil microemulsions (Figure 1 6 C ) 79 Even though the size of water in oil microemulsions can be tuned by the volume ratio of water and oil, surfactant type, and reaction temperature, MNP s generated by microemulsion usually have a bro ad size and shape distribution. 66 In addition, the yield of nanoparticles is relatively low compared to co precipita tion and thermal decomposition. 66 Moreover, large amounts of solvents are needed to produce appreciable amounts of nanoparticles, which makes micr oemulsion ineffic ient and difficult to perform in large amounts 66 Surface engineering of magnetic nanoparticles In order for MNP s, especially the hydrophobic MNPs described above to be used for various bioanalytical an d biomedical applications, the ability to provide them with desired solubility and targeting in biological fluids without aggregation is a critical step. Typical surface engineering methods for MNP s are ligand exchange, 80 81 ligand modification, 82 and surface coating 83 87 In ligand exchange, the original hydrophobic surfactants are replaced with h ydrophilic ligands which occur s when the hydrophilic ligands have higher affinity to the MNP s than the original hydrophobic surfactants. Typical examples include t he use of dopamine derivatives 80 and dimercaptosuccinic acid (DMSA) (Figure 1 7 A ) 81 Dopamine derivatives contain a bidentate enediol structure that can convert the under coordinated Fe surface sites back to a bulk like lattice structure with an octahedral geom etry for
32 oxygen coordinated Fe resulting in their tight binding to iron oxide nanoparticles. 80 88 DMSA first forms a stable coating onto the MNP surface through carboxylate chelate bonding and further stabilizes the ligand shell by forming intermolecular disulfide cro ss linkages among the ligands. 81 Ligand modification refers to the proces s of changing the original hydrophobic surfactants to hydrophilic ligands through chemical reactions. For example, oleylamines and oleic acids on the surface of MNP s can be oxidized into azelaic acids, generating free carboxylic acid groups that confer hig h solubility in aqueous solutions and allow further conjugation with biomolecules (Figure 1 7 B ) 82 Surface coating of MNP s can also be rea lized by encapsulating them in a hydrophilic lay er. This layer can be polymer, 83 silica, 84 85 metal, 86 or others (Figure 1 7 C ) 87 When polymers are used, these polyme rs are usually amphiphilic (e.g., phospholipid PEG), with their hydrophobic part interacting with hydrophobic surfactants on the surface of MNP s and their hydrophilic part on th e exterior to render modified nanoparticles with desired solubility in aqueous solutions. Silica coating prevents engineered MNP s from aggr egation in two different ways. 89 One is by shielding the magnetic dipole interaction with the silica shell. On the other hand, silica coated MNP s are n egatively charged. Recently, surface coating of MNP s with other ino rganic layers, including metal s 86 and others, 87 are also emerging. Applications of magnetic nanoparticles In the past decade s the synthesis and surface engineering of MNP s have been i ntensively investigated not only for their fundamental scientific interest but also for their technological applications, including magnetic storage, biosensor construction, and nanomedicine development, etc This section provides a more specific review of MNP
33 use for magnetic resonance imaging (MRI), biological separation, drug delivery, and hyperthermia therapy. MRI is one of the most promising applications for MNP s in vitro as well as in vivo By grafting a synthetic mimetic of sialyl Lewisx (sLex), a n atural ligand specific for endothelial inflammatory adhesion molecule E selectin expressed on leukocytes, on dextran coated ultrasmall iron oxide nanoparticles, targeting of leukocytes by MRI was successfully performed with the resulting MNP s in cell cultu re and mouse model (Figure 1 8 A ) 90 Recently, MNP s have also been designed to detect molecular interactions in biological media after functionalizing biomole cules onto their surfaces. 91 92 In the presence of a biological target, the surface conjugated biomolecules induce either cooperative agg regation or dispersion of the nanosensors, changing the spin spin relaxation time, T 2 of water molecules that can be detected by magnetic relaxation measurements or MRI. These nanosensors have been us ed to detect oligonucleotides, 91 proteins, 91 and cells 92 with high sensitivity. In addition, measurements can even be conducted in turbid media, cell lysate, and whole blood, which is normally not feasible for absorption or fluorescence measurements. 9 1 Another important application of MNP s is their use for biological separation. Compared to traditional separation methods, magnetic separation is fairly simple. In addition, all steps of the separation can take place in one tube without expensive liquid chromatography systems. 93 Fan et al. have developed biotinylated M NP s for fluorescently labeled avidin iso lation. 94 Using nitriloacetic acid coated magnetic nano particles, Xu et al. have separated histidine tagged proteins from cell lysate upon
34 chelation to Ni 2+ (Figure 1 8 B ) 8 0 In addition, MNP s have also been conjugated with antibodies to capture circulating tumo r cells (CTCs) from blood. 95 MNP s can also be used as carrie rs fo r drug delivery. Researchers have found that MNP s with diameter s ranging from 10 to 100 nm are optimal for intravenous injection and have the most prolonged blood circulation times. 96 These nanoparticles are small enough to evade the reticuloend othelial system (RES) and to penetrate small tissue capillaries, offering the most effec tive dis tribution in targeted tissues. 96 When using MNP s for drug delivery, effective targeting, which helps avoid undesired side effect s, can be achieved b y use of an external magnetic field, 97 pa ssive targeting, 98 active targeting, 99 alone or in combination 100 According to the re sults obtained by researchers a strong magnetic field gradient at the tumor site can induce the accumulat ion of MNP s (Figure 1 8 C ) 101 102 Due to the enhanced permeability and retention (EPR) effect, MNP s with proper sizes ca n gather around tumor tissues. 98 Also, by engineering MNP s wit h targeting ligands that promote cell specific interactions (e.g., aptamers), they can selectively bind to certain cancer cells. 99 Thro ugh electrostatic interaction or covalent binding, MNP s have been widely used for the delivery of different kinds of anticancer drugs. Using MNP s, Gallo et al. have even constructed a platform which possesses the capability to cross the blood brain barrier to deliver oxantrazole to the brain. 103 The application of MNP s for hyperthermia therapy was first envisi oned in 1993 by Jordan et al., 104 who demonstrated that superparamagnetic n anoparticles could efficiently convert the energy of an oscillating m agnetic field to heat. 104 Due to the fact that tumor cells are more sensitive to temperatu re increase than healthy cells, this
35 property can be used in vivo to kill tumor cells by hyperthermia therapy (Figure 1 8 D ) 102 105 Preclinical and clinical data have shown that hyperthe rmia therapy is feasible. In addition, it is very effective when combined with radiation therapy. According to a recent study, the survival rate of patients with glioblastoma multiforma doubled when radiation therapy was coupled with hyperthermia thera py 106 which allows an increase of perfusion in the tumor tissue and therefore a higher oxygen content making the ra diation therapy more powerful. 107 Graphene Oxide s Graphene oxide s (GO s), two dimensional carbon nanomaterial s, have been intensively investi gated for their unique properties and potential applications in transparent conductors, ultrafast transistors, and nanocomposite materials. These fascinating properties of GO s are mainly derived from their distinct chemical structure s composed of small sp 2 carbon domains surrounded by sp 3 carbon domains and oxygen containing hydrophilic functional groups. 108 Recently, the understanding of various physical and chemical properties of GO s has expanded their applications toward bioanalytical and biomedical areas Because of their excellent amphiphilicity, aqueous solubility, surface functio nalizability, and fluorescence quenching ability, GO s are promising candid ate s for biological applications. 108 This section review s recent efforts to apply GO s to bianalytical and biomedical research. Many GO based biosensing platforms have been based on the preferential interaction of GO with single stranded DNA (ssDNA) over double stranded DNA (dsDNA) or aptamer target complex (Figure 1 9 A ) 109 The exposed nucleobases in ssDNA are strongly adsorbed on GO surface through interactions, whereas nucleobases in dsDNA or aptamer target complex are effectively hidden in helical or
36 tertiary structure s Therefore, when fluorescently labeled ssDNA probes on the GO surface are hybridized or bound to their targets, the fluorescen ce of the dye quenched by GO s will be res t o red. Using this principle, sensitive detection of various nucleic acid ( e.g., mRNA, microRNA ) 110 111 and non nucleic acid ( e.g., ATP, toxin ) 112 113 targets in buffer systems has bee n realized. In addition, the limit of detection has been further improved by several signal amplification stragegies. 111 Inspired by the research developments of using fun ctionalized carbon nanotubes for macromolecule delivery, Lin et al. ha ve successfully transferred GO based sensing platforms from buffer systems to living cells. According to their results, G O s are good vehicle s to transport nucleic acid probes into living cells, protecting the loaded nucleic acid probes from enzymatic digestion and enabling in situ molecular imaging. 114 At this time ATP, 114 115 microRNA, 116 and histidine tagged protein 117 detection in living cells has been achieved. To refine the perf ormance of the GO based biosensing platforms, it is important to understand the molecular mechanism s of GO F ster resonance energy transfer ( FRET ) as well as to investigate the influencing factors of the process. To this end, researchers have systematical ly studied the impact of GO reduction degree 118 and ssDNA sequence length 119 on GO FRET biosensor behavior. B ecause of their high efficiency in absorbing and transferring UV laser energy to analyte, GO s ha ve been considered as candidate s t o replace conventional organic matrices for laser desorption/ionization mass spectrometry ( LDI MS ) The high efficiency of GO s as LDI MS matrices is attributed to their high heat dissipation and electron transfer properties. 120 For molecules contain ing aromatic structures, GO s can
37 serve as non specific affinity probe s for LDI MS analysis. 121 When conjugated with apt amers, the corresponding conjugates can serve as specific affinity probe s for LDI MS analysis (Figure 1 9 B ) 122 To further increase the efficiency of GO s as LDI MS matrices both multiwall carbon nanotubes (MWCNTs) 123 and gold nanoparticles (AuNPs) 124 have been introduced onto GO s to construct hybrid nan o film s which showed excellent applicability for LDI MS analysis of small molecules, enzymes, and tissues. In addition, the efficiency of the hybrid nanofilm could be improved more by enhancing the surface roughness and thicknes s. 125 GO s can be used as delivery vehicle s for drug, gene, and other therapeutic mo dalities owing to their high loading capacities Many small molecule drugs with pH dependent solubility, such as doxoribucin (DOX) 126 captothecin (CPT), 127 ibuprofen, 128 and 5 fluorouracil, 128 can be delivered into living cells by GO s and released in a pH responsive manner. When GO s are used as carrier s for gene s polyethyleneimine (PEI) has been widely emplo yed as a surface modifier In this manner gen etic materials, including siRNA 129 and pDNA (Figure 1 9 C ) 130 can be loaded onto GO s through electrostatic interaction s or covalent conjugation. Recently, the potential of chitosan for GO surface modific ation has been investigated 131 To enhance therapeutic efficacy through combinational therapy, photosensitizer s have also been loaded together with anticancer drug s for combined chemotherapy and photodynamic therapy. 132 In addition, GO/TiO 2 has been loaded with anticancer drug for combined chemotherapy and photocatalytic therapy. 133
38 In addition to applications as delivery carrier, GO s have been utilized for photothermal therapy based o n their high absorption of near infrared ( NIR ) laser radiation For example, high therapeu tic efficacy of PEGylated GO s ha s been demonstrated in mouse tumor xenograft models (Figure 1 9 D ) 134 Recently, it has been found that the photothermal effect of GO s originates from its ability to induce oxidative stress, mitochondrial depolarizati on, and capase activation, leading to apoptotic and necrotic cell death. 135 DNA Micelles Micelles, including normal phase (oil in water) and inverse (water in oil) micelles, are aggreg ates of amphiphilic molecules dispersed in liqui d colloids. Typical micelles in aqueous solutions form a ggregates with the hydrophilic head in contact with surrounding solvent sequestering the hydrophobic tail in the micell e center. When the hydrophilic h ead is composed of DNA sequences, corresponding micelles are called DNA micelles. DNA as a unique biomolecu le provides several advantages The availability of fully automa ted solid phase synthesis can provide DNA sequences with nucleotide precision and tun able modifications, thereby facilitating the fabrication of monodispersed nanomaterial s with predictable properties. 136 The powerful molecular recognition of DNA through either base pairing or target induced folding is anothe r key feature of DNA micelles. 137 Furthermore, the small size, repeated structure, and suitable stiffness of DNA sequences play a key rol e in bottom up nanotechnology. 4 The hydrophobic tail of DNA micelles can be block c opolymers or lipid molecules. This section fo cus es on the synthesis and application s of DNA micelles
39 Synthesis of DNA micelles DNA micelles can be synthesized through chemical couplings or molecular biology based approaches. Coupling in solution is relatively easy and ca n be readily realized by amide 138 or disulfide 139 bon d formation, Michael addition, 140 as well as copper cat alyzed Huisgen cyclo addition. 141 When using copper catalyzed Huisgen cyclo addition, the introduction of a copper chelating reagent is desired since Cu 2+ can induce DNA strand breaks. 141 However, due to the distinct solubility difference between DNA molecules and block copolymers or lipid molecules, the yield of solution coupling is fairly low and careful selection of bridging solvent is required. 136 For these reasons, coupling strategies based on solid phase synthesis have been explored and developed. The first trial in this direction was perfo rmed by Mirkin et al. in 2004. 142 The key reagent in this approach was a polystyrene phosphoramidite, which was obtained by treating hydroxyl terminated polystyrene with chlorophosphoramidite. DNA sequences were prepared through solid ph ase synthesis on an automated DNA/RNA synthesizer and the coupling of polystyrene phosphoramidite to them was carried out usin g a syringe synthesis technique. 142 This method has some drawbacks since reproducible and efficient exposure of the polymer phosphoramidite to the solid phase is not guaranteed. 136 To overcome these deficiencies, Herrmann et al. esta end of DNA sequences on a con ventional DNA/RNA synthesizer. 143 The advantages of their approach lie in the high reproducibility due to automation and the efficient exposure of polymer phosphoramidite to the solid phase. In addition, the coupl ing of la rge polymer phosphora midites has
40 remarkably high yields, reaching up to 41% and 32% for PPO (poly(p phenylene oxide)) polymers with molecular weights of 1000 and 6800 g/mol, respectively. 143 defined DNA micelle system usi ng diacyllipid DNA conjugates, which can be simply and efficiently synthesize end of DNA sequences on an automated DNA/RNA synthesizer wit h an even higher yield of 60%. 144 Compared to DNA micelles with block copolymer tail, these are fairly uniform, as the block copolymer has a wide molecular weight distribution and the diacyllipid molecule only has fixed molecular weight. 142 In addition, DNA micelles with diacyllipid tail have excellent thermal stability and low critical micelle concentration (CMC), with intact molecular recognition of DNA. More i mportantly, these DNA micelles possess size dependent perme ability into living cells, 137 144 making them excellen t candidates for intracellular bioanalysis. Besides chemical couplings, molecular biology based approach is another important tool for the preparation and postsynthetic modification of DNA micelles. Due to the cumulative yield limit of solid phase synthesi s, the length of DNA sequences in DNA micelles is restrained to 100 nucleotides. To synthesize DNA micelles with longer DNA sequences, molecular biology techniques, suc h as PCR 145 enzyme restr iction, 146 and enzyme ligation, 146 have been employed. Using PCR, DNA micelles with DNA sequences having u p to 1000 bases can be generated. To obtain DNA micelles with DNA sequences contain several thousand bases, the use of restriction and ligation enzymes is needed.
41 Applications of DNA micelles Enabled by their supramolecular self assembl y propertie s, DNA micelles have been applied to the delivery of anticancer drugs, 137 147 as 3D scaffolds for organic reactions, 143 the regulation of target genes, 45 148 and as combinational tools for cancer therapy. 45 149 150 DNA micelles have been successfully tested in vitro as anticancer drug delivery vehicles, with the distinct advantage of increasing both the so lubility and stability of hydrophobic pharmaceutical s. 144 With folic acid as the targeting ligand, Herrmann et al. achieved selective killing of Caco 2 cells (cancerous human colon adenocarcinoma cells with over expression of folat e re ceptors) using DOX loaded DNA micelles. 147 In unction but it enables the convenient incorporation of folic acid. To fully utilize the DNA shell of DNA micelles, we have developed an aptamer micelle nanostructure by covalently linking a diacy llipid onto aptamer sequences (Figure 1 10 A ) 137 Because of the aptamer shell, aptamer micelles with built in molecular recognition have strong specific binding to target cancer cells at physiological conditions. Using a model drug, we have proved the feasibility of using aptamer micelles for targeted drug delivery in culture media and blood vessel s Taking advantage of the remarkably precise molecul ar recognition of DNA, researcher s have recently started to explore the potential of using it as programmable nanoreactor (Figure 1 10 B ) 151 152 DNA templated chemistry allows defined spatial arrangement of DNA bound reaction partners. Due to the close proximity of the reactive groups in the micelle confinement, several organic reactions, including isoindole formation, Michael addition, and amide bond formation, have been carried out using
42 DNA micelles. 143 For these DNA micelle templated reactions, the specific interaction of micelle DNA with reactant DNA is crucial. Other than serving as building blocks for DNA micelles to provide them with desired molecular recognition capabilities, DNA molecules can also be tested for treating various genetic diseases. Antisense DNA and RNA have been used to block the expression of specific proteins, especially deleterious ones, by hybridizati on with target mRNA sequences. 153 Taking advantage of this property Park et al. have explored the potential use of DNA micelles for the delivery of antisense DNA (Figure 1 10 C ) 148 These DNA mi celles were assembled from poly (D,L lactic co glycolic acid) (PLGA) conjugated DNA. Compared to free DNA molecules, these DNA micelle s can be more efficiently transported into living cells, where DNA molecules complementary to target mRNA can be released in a sustained manner by controlled degradation of PLGA. However, Park et al. d DNA molecules. To push this line of research further, we have recently constructed a DNA micelle system whose DNA shell can serve as a potent tool for knocking down ocogene, leading to the apo ptosis of target cancer cells. 45 Other than these applications, DNA micelles are excellent combinational tools for effective cancer therapy. For example, we have designed and synthesized a multifunctional DNA micelle system with sensitive intracellular imaging and effective gene regulation. 45 In addition, we have found that DNA micelles can also be used for in vivo cell modification and localized immunotherapy. 149 150 There are two approaches to achieve immunot herapy using DNA micelles. One is the use of DNA micelles for the delivery of immunomodulators. 149 Typical examples include u nmethylated cytosine
43 guanosine mot ifs (CpG) that mimic molecular sign atures of pathogens and trigger an immunostimulatory cascade including maturation, differentiation, and proliferation of multiple host immune cells through pattern recognition receptors. 154 The other approach is the use of DNA micelles t o bring immune effector cells (e.g., cells that naturally attack cancer cells 155 and cells that can be genetically engineered to produce therapeutic s in situ after being delivered 156 ) into close proximity of target cancer cells (Figure 1 10 D ) 150
44 Figure 1 1. The composition and structure of nucleic acids. A ) The composition of nucleic acids. A nucleotide consists of a phospha te group, a sugar, and a nitrogen containing base. The sugars have two different chemical forms: ribose (in RNA) and deoxyribose (in DNA). The nitrogenous bases also have two different chemical forms: pyrimidines, including cytosine (C), thymine (T), and u racil (U), as well as purines, including adenine (A) and guanine (G). B ) The structure of nucleic ac ids using DNA as an example. Nucleotides join together in a series to form a nucleic acid ; the 5 end of one nucleotide attaches to the 3 end of the adjace nt nucleotide through a phosphodiester bond When two complementary nucleic acids are hybridi zed the ir sugar phosphate ends are arraged in opposite directions forming a doule helical structure
45 Figure 1 2. Chemical synthesis of nucleic acids. A typica l cycle of the chemical synthesis of nucleic acids includes four steps: deprotection, coupling, capping, and stabilization
46 Figure 1 3. Representative base modifications for nucleic acids. 14 Figure 1 4. Schematic il lustration of cell based SELEX. DNA sequences that have s pecific binding to target cells but not to control cells, are evolved to enrich the selection pools. The enriched pools are then clon ed and sequenced to indentify aptamer candidates. 29 (SELEX = Syste matic Evolution of Ligands by E X ponential e nrichment )
47 Figure 1 5. Working p rinciple of MBs and AMBs A) For MB s, the hybridization between them and the co mplementary nu cleic acid target physically and spatially separates the fluorophore and the quencher to restore fluorescence. B) For AMBs the separation of the fluorophore and the quencher is achieved through the binding between the aptamers and the non nucleic acid tar gets. 41 (MB = molecular beacon, AMB = aptamer molecular beacon) Figure 1 6. Schematic illustration of typical me thods for MNP synthesis. A ) Co precipitation using Fe 3 O 4 nanoparticles as an e xample; B ) Thermal decomposition, includ ing growth mechanism and set up; C ) Microemulsion. (MNP = magnetic nanoparticle)
48 Figure 1 7. Surface engineeri ng of MNPs A ) L igand exchange by dopamine deriv at ive and dimercaptosuccinic acid; B ) Ligand modifica ti on using ligand oxidation as an example; C ) Surface coating. The coating layer can be polymer, silica, metal, and others. (MNP = magnetic nanoparticle)
49 Figure 1 8. Applications of MNPs A ) Magnetic resonance imaging ; 90 B ) B iological separation using histidine tagge d protein separation as an example ; 8 0 C ) D rug delivery ; D ) H yperthermia therapy. For C ): Gray spheres are MNP s and or an g e sp heres are drug molecules. For D ): AMF = alternating magnetic field. 102 (MNP = magnetic nanoparticle)
50 Figure 1 9 Applications of GO s. A ) Biosensor development ; 109 B ) Laser desorption/ionization mass spectrometry ; 122 C ) Delivery vehicle u sing PEI/G O complex for gene delivery as an example ; 157 D ) Hyperthermia therapy. 134 (GO = graphene oxide, GO/PEI = graphene oxide/p olyethylenimine )
51 Figure 1 10. Applications of DNA micelles. A ) Drug delivery ; 137 B ) 3D scaffolds for organic reactions ; 151 152 C ) Gene regulation ; 148 D ) C ombinational tools for cancer therapy 150
52 CHAPTER 2 SMART MULTIFUNCTIONAL NANOSTRUCTURE FOR TARGETED CANCER CHEMOTHERAPY AND MAGNETIC RESONANCE IMAGING Background Since most anticancer drugs are unable to differentiate between diseased and healthy cells, systemic toxicity and undesired side effects can result. 158 However, these issues can be addressed through tumor s pecific delivery of anticancer drugs using nanostructures equipped with targeting moieties. 159 Many tumors possess fenestrated vasculature and poor lymphatic drainage because of their rapid growth. At the same time, an enhanced permeability and retention (EPR) effect results, 160 allowing nanostructures to accumulate specifically at the tumor site by the mechanism of passive targeting. In addition, tumor localization of nanostructures can be further enhanced by active targeting groups that have mo lecular recognition to target cancer cells, such as the vitamin folic acid, 161 162 peptides, 163 164 and aptamers. 137 165 170 However, even if a high level of anticancer drugs can reach the tumors, this does not mean that sufficient drug molecules will automatically be taken up by the target cancer cells to effectively kill them. 171 172 Therefore, other than tumor targeting, the ability to induce efficient uptake of anti cancer drugs is another important factor to consider when choosing the proper targeting ligand. 173 Aptamers are single stranded oligon ucleotides that can bind specifically to their targets, which range from small molecules to proteins, whole cells, and even tissues, by folding into distinct secon dary or tertiary structures. 170 Generated by a process called SELEX (Systematic Evolution of Ligands by EXponential enrichment) developed in the Targeted Cancer Chemotherapy and Magnetic Resonance Imaging, ACS Nano 5 7866
53 1990s, 21 22 aptamers rival antibodies for their molecular recognition abili ty. 174 Thus, they have emerged as new targeting moieties for biotechnological and therapeutic applications. Recently, our group has developed a cell based SELEX strategy that can produce a panel of aptamers for cancer cells. 31 175 In addition to specific binding to cancer cells, some of these aptamers can be internalized into cancer cells, 176 thus making them good candidates for intracellular drug delivery. Among all nanocarriers, smart nanostructures that are responsive to external stimuli, such as heat, 177 light, 178 or acidic conditions, 179 have attracted the attention of researchers. By formulating these smart nanostructures with imaging contrast agents, an all in one system combini ng tumor targeting, tumor therapy, and tumor imaging is possible. 180 182 In this study, a smart multifunctional nanostructure (SMN) was constructed of three c omponents: a porous hollow magnetite nanoparticle (PHMNP) as a carrier, a heterobifunctional PEG ligand as a linker, and an aptamer as a targeting moiety. This nanostructure was successfully utilized for targeted chemotherapy and magnetic resonance imaging (MRI). The hollow interior of the PHMNPs was loaded with the anticancer drug doxorubicin (DOX). Multiple aptamers on the outer layer of SMN resulted in a multivalent effect, leading to enhanced specific binding and internalization of SMNs to target cancer cells. Because of having acid labile pores, upon arrival of lysosomes, the acidic environment of the lysosomes facilitated the release of DOX from SMN, enabling efficient killing of target cancer cells. In addition, T 2 relaxation measurements and T 2 weig hted MRI images showed that this nanostructure had great potential to be used as a T 2 contrast agent.
54 Experimental Section Synth esis of Heterobifunctional PEG L igand The synthesis of h eterobifunctional PEG ligand is described as follows (Figure 2 1): Synth esis of 3, 4 d ihydroxyhydrocinnamic pentafluorophenol ester (1): 3,4 d ihydroxyhydrocinnamic acid (1.82 g, 10 mmole) and pentafluorophenol (2.208 g, 12 mmo le) were dissolved in 40 mL 1,4 dioxane in a 150 mL round bottomed flask connected to a dropping funne l under d icyclohexylcarbodiimide (DCC) (2.06 g, 10 mmole) in 10 mL 1,4 dioxane was then added dropwise. The solution was stirred overnight under Ar at room temperature. After the precipitate was filtered from the solution, the solvent was removed using a rotary evaporator. The remaining oily product was purified by silica gel column chromatography with hexane/ethyl acetate (V: V = 3:1) to yield a yellow oily product. 1 H NMR (300 MHz, CDCl 3 8.1 Hz),6.76 (1H, d,J = 1.5 Hz), 6.66 (1H, dd, J =8.1, 1.5 Hz), 2.94 (4H, m) Synthesis of heterobifunctional PEG ligand with active amino and carboxyl groups (NH 2 PEG COOH) (2): b is(2 aminopropyl) polypropylene glycol block poly ethylene glycol block polypropylene glycol (Mw = 1900, 9.5 g, 5 mmole) was dissolved in 200 mL anhydrous d imethylformamide ( DMF ) in a 500 mL round bottomed flask connected to a dropping funnel under a blanket of Ar. Triethylamine (TEA) (696 L, 5 mmole) an d succinic anhydride (0.6 g, 6 mmole) in 25 mL anhydrous DMF were then added dropwise. The solution was stirred 24 h under Ar at room temperature. The solvent was then removed using a rotary evaporator. The remaining oily product was redissolved in 15 mL m ethylene chloride and precipitated by adding 150 mL ether under dry ice. The precipitation procedure was repeated 3 times. Finally, the light yellow
55 product was obtained by filtration and dried by a lyophilizer. The product was identified by 1 H NMR accordi ng to da ta reported in the literature. 183 Synthesis of heterobifunctional PEG ligand with catechol group and carboxyl group (DPA PEG COOH) (3): 3,4 d ihydroxyhydrocinnamic pentafluorophenol ester (0.4176 g, 1.2 mmole), NH 2 PEG COOH (2 .1 g, 1 mmole) and TEA (139 L, 1 mmole) were dissolved in 20 mL methylene chloride and stirred overnight under Ar at room temperature. The solvent was removed using a rotary evaporator. The remaining oily product was redissolved in 3 mL methylene chloride and precipitated by adding 30 mL ether under dry ice. The precipitation procedure was repeated 3 times. Finally, the light yellow product was obtained by filtration and dried by a lyophilizer. The product was identified by 1 H NMR according to da ta reporte d in the literature. 183 PEGylation of Porous Hollow Magnetite Nanoparticles and Doxorubicin loading A 5 mg sample of PHMNPs dissolved in 5 mL of tetrahydrofuran (THF) was degassed under a blanket of Ar for 15 min and transferred to a dropping funnel. The solution was added dropwise into 75 mg of heterobifunctional PEG ligand dissolved in 15 mL of THF over a period of 6 h under Ar The resultant mixture was stirred under a blanket of Ar at 50 C overnight. The P EGylated porous hollow m agnetite nanoparticles ( PPHMNPs ) were then precipitated by adding hexane and collected by a strong magnet. The collected nanoparticles were w phosphate buffered saline ( PBS ) three times and then dispersed in ult rapure water or PBS for f urther use. For DOX loading, a similar experimental procedure was used, but 10 mg of DOX was premixed with 75 mg of heterobifunctionl PEG ligand dissolved in 15 mL of THF. In addition, after the reaction, the solvent was evaporated under low pressure.
56 Synt hesis of Aptamer The sgc8 aptamers with amino group and desired dye modificat ions were synthesized on an ABI 3400 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA, USA). The aptamer with f luorescein isothiocyanate ( FITC ) labeling was used primarily for flow cytometry, while the aptamer with c arboxytetramethylrhodamine ( TAMRA ) labeling was used for confocal fluorescence microscopy. The aptamer without dye labeling was used for the cytotoxicity assay. Detailed sequence information is provided in Table 2 1 The aptamers with and without FITC labeling were deprotected in 3 mL of AMA solution (ammonium hydroxide : 40% aqueous methylamine = 1 : 1) at 65 C for 25 min. The TAMRA labeled aptamers were deprotected in 3 mL of TAMRA deprotection solution (metha nol : tert butylamine : water = 1 : 1 : 2) at 65 C for 4 h. All cold ethanol. Then the precipitated aptamers were collected by centrifugation and hylammonium acetate (TEAA) for further purification by reversed phase high pressure liquid chromatography (RP HPLC) (ProStar, Varian, Walnut Creek, CA, USA) using a C18 column and acetonitrile TEAA solvent. Finally, these aptamers were quantified by measur ing their absorbance at 260 nm. Anchoring of Aptamers onto P EGylated Porous Hollow Magnetite Nanoparticles To immobilize aptamers onto PPHMNPs, standard peptide bond formation methodology was used. To with a concentration of 1.5 ethyl 3 (3 dimethylaminopr opyl) carbodiimide (EDC) in PBS The resultant mixture was incubated at room temperature for 15 min hydrox ysuccinimide (NHS) in PBS L of 50 were added and further incubated at room
57 temperature for another 1 h. Successfully synthesized SMNs were collected by a strong magnet and washed three Fina lly, the resultant product was e ither redispersed in PBS Doxorubicin Payload Determination and Release Kinetics Study The DOX payload determination and release kinetics study were carried out using dialy sis membrane tubing (MWCO = 3500). The released DOX can cross the dialysis membrane, but not the SMNs. In order to determine the maximal loading of DOX into PHMNPs, 2 mg of DOX loaded PPHMNPs d ispersed in 0.5 mL of PBS in dialysis membrane tubing wa s float ed in 20 mL of PBS (pH = 7.4) at 37 C. After 72 h, a 2 measured. In order to study the release kinetics of DOX from PPHMNPs, a similar experimental setup with the same temperature and releas e time was used, but PBS with differen aliquot was replaced to maintain the total volume. Binding Test To demonstrate t he specific targeting of aptamer and SMNs toward different cell lines, fluorescence measurements were obtained on a FACScan cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). A green laser at 488 nm with different excitation voltages (650, 700, and 750 V) was used as the excitation source. Samples containing CEM cells or Ramos cells with a concentration of 10 6 cells/mL were incubated with the desired concentrations of aptamer or SMN s on ice in a
58 afte r flow cytometry measurement was then directly subjected to confocal fluorescence microscopy. Internalization and Co localization Study To investigate the internalization of aptamers and SMNs into different cell lines, samples containing CEM cells or Ramos cells with a concentration of 10 6 cells/mL were incubated with the desired concentrations of aptamer or SMN at 37 C in a volume of 2 atmosphere. The cells were then centrifuged, L of binding buffer, and subjected to confocal fluorescence microscopy analysis using an Olympus FV 500 IX81 confocal microscope (Olympus, Center Valley, PA, USA) having a 40 oil dispersion objective. A 488 nm Ar laser was the excitation source for FITC d ye, and a 543 nm Ar laser was used for the excitation of TAMRA dye. For the co localization study, 10 M lysosensor was added for specific staining of the lysosomes of cancer cells during the last 0.5 h of the 2 h incubation. The remaining experimental pro cedures were the same as those for the internalization study. Cytotoxicity Assay The cytotoxicity of SMNs only, DOX only and DOX SMNs to CEM cells and Ramos cells was evaluated using the CellTiter 96 proliferation assay (Promega, Madison, WI, USA). A samp le of 1 10 5 seeded into each test well on a 96 well plate. Then SMNs only, DOX only (0 and DOX SMNs resultant cell mixture was incubated at 37 C in a 5% CO 2 atmosphere for 2 h. Then, 75
59 well plate was then put back into the incubator for another 48 h. Fi well, and the 96 well plate was subjected to absorption measurement at 490 nm using a VersaMax tunable miroplate reader (Molecular Devices, Inc., Sunnyvale, CA). Relaxation Measurements and Magnetic Res onance Imaging T 2 relaxation measurements were carried out at 1.4 T using a standard Carr Purcell Meiboom Gill (CPMG) sequence on a benchtop Minispec mq60 time domain nuclear magnetic resonance ( TD NMR ) contrast agent analyzer (Bruker Optics, Billerica, MA USA). SMNs only, SMNs with CEM cells, and SMNs with Ramos cells were incubated on ice fo in flow tubes. The final 6 cells/mL, respectively. In order to address the concern of SMN aggregation and settling, samples were vortexed for more than 30 s. Then, all the samp les were directly transferred into the NMR sample tubes and subjected to T 2 relaxation measurements, without any further washing steps. T 2 weighted MRI images were taken on a 11 T/470 MHz MRI spectrometer (Bruker Optics, Billerica, MA). SMNs only, SMNs wi th CEM cells, and SMNs with Ramos cells were incubated on ice fo r 30 min in 50 in 1.5 mL Eppendorf tubes. The final cells was the same as in the T 2 relaxation measurements. After incubation, the samples in Eppendorf tubes were vortexed, fixed on a homemade foam sample holder in a 4 3 array, and then put in the coil. T 2 weighted MRI images were acquired with a gradient echo sequence (TR = 4000 ms and TE = 20.4 ms).
60 Results and Discussion Synth esis and Characterization of Smart Multifun ctional Nanostructure s SMNs were prepared as outlined in Figure 2 2 A First of all, PHMNPs were obtained through a th ree step reaction: 183 First, 13 nm iron magnetite core shell nanoparticles (IMNPs) ( Figure 2 2 B ) were produced by th ermal decomposition of iron pentacarbonyl (Fe(CO) 5 ). Second, through controlled oxidation of IMNPs in the presence of the oxygen transfer reagent trimethylamine N oxide (Me 3 NO), 16 nm hollow magnetite nanoparticles (HMNPs) were obtained. The transmission e lectron microscopy (TEM) image ( Figure 2 2 C ) showed that they have hollow interiors about 10 nm in diameter and magnetite shells around 3 nm in thickness. Third, acid etching of HMNPs in the presence of oleic acid at high temperature resulted in PHMNPs ( Fi gure 2 2 D ). The hollow interiors of PHMNPs were encircled with discrete polycrystalline magnetite domains, and the pores of the PHMNPs were 2 4 nm. The as prepared PHMNPs are hydrophobic by their oleylamine/oleate coating and have no active functional grou ps. Therefore, the heterobifunctional PEG ligand with a catechol group on one end and a carboxyl group on the other end was synthesized according to a method reported in the litera ture 183 with proper modifications ( see Experimental S ection for detailed synthesis route ). The ligands were introduced onto the surface of PHMNPs to make them hydrophilic through ligand displacement. The resultant PPHMNPs were easily dispersed in aqueous solution as shown in Figure 2 2 E and had a hydrodynami c diameter of around 60 nm. In addition, the heterobifunctional PEG ligand also equipped PPHMNPs with an active carboxyl functional group. At last, aptamers with an amino group and a desired fluorophore modification were prepared through solid phase synthe sis using an automatic DNA /RNA synthesizer
61 and anchored onto the surface of PPHMNPs by reacting with the active carboxyl group on the heterobifunctional PEG ligand. In order to avoid any adverse effect on binding specificity and affinity, 10 thymine (T) ba ses were inserted between the amino group and the aptamer sequence. PPHMNPs do not show any fluorescence; however, a strong fluorescence signal was observed for SMNs after the successful introduction of fluorophore labeled aptamers. Figure 2 2 F shows an ex ample of usi ng TAMRA labeled aptamers. TAMRA is a commonly used fluoreophore modifier for DNA sequences with emission around 580 nm when excited around 540 nm. Doxorubicin Loading and Release Profile Figure 2 3 shows the mechanism of using SMNs for targete d cancer chemotherapy. The anticancer drug DOX was loaded into the hollow cavity of SMNs. Owing to aptamers on the surface of DOX SMNs, the loaded nanoparticles can specifically bind and then enter target cancer cells through receptor mediated endocytosis. Since the pores of SMNs are stable at physiological pH but vulnerable to acidic pH, the low lysosomal pH level will enlarge the pores of SMNs, accelerating the release of DOX from SMNs and the killing of target cancer cells. In order to have a large loadi ng capacity, drug loading and PEGylation of PHMNPs were performed simultaneously to reduce the barrier effect to a minimal extent. As quantified by measuring the absorbance of DOX at 485 nm, the drug payload was 8.72 wt %. For investigating the release kin etics of DOX loaded into PPHMNPs, the particles were dialyzed against PBS at different pH values (pH = 5, 6, and 7.4). The release of DOX was shown to be a pH dependent, diffusion controlled process ( Figure 2 4 ). At physiological pH (pH = 7.4), a gradual i ncrease and a plateau were observed after the initial burst of DOX release. The t 1/2 (the time needed for the release of 50% of
62 the maximal loading) was approximately 21.5 h. For lower pH values, a similar release behavior was observed, albeit with shorter t 1/2 (7.5 h at pH = 6 and 3.5 h at pH = 5). We attribute the observed pH dependent release behavior to the low pH environments, which can enlarge the acid labile pores of PHMNPs to accelerate the rate of DOX release from DOX loaded PPHMNPs. 183 Thus, the lower the pH value, the faster the release of DOX. Since the oleylamine/oleate layer on the as prepared PHMNPs is successfully replaced with a layer of the heterobifunctional PEG ligand through ligand displacement as mentioned before, the possibility of DOX release at acidic pHs due to oleic acid protonation can be excluded. In addition, because of the concrete structure of PHMNPs, the probability of DOX entering the nanoparticle matrix during its loading process is very low, and thus it is unlikely that DOX would release from the nanoparticle itself under acidic environments. Although the nitrogen atoms in the heterobifunctional ligand may be protonated at low pH values to alternate the surface charge of PPHMNPs, we believe that this i s unlikely to be the reason for DOX release since it is already loaded into the cavity of PPHMNPs. However, the pH dependent solubility of DOX 184 185 may also play a role in the release kinetics other than acid etching of the pores. The reported pH in lysosomes 186 188 is between 4.0 and 6.5, which is relatively low compared to the physiological pH of 7.4. Therefore, the release of DOX from SMNs can be achieved inside the lysosomes. Binding and Internalization behavior of Smart Multifunctional Nanostructure s In order to determine if the conjugated aptamer still preserves its binding affinity and specificity, the binding of SMNs toward target cancer cells and control cancer cells was studied using flow cytomet ry. FITC labeled sgc8 which specifically binds to cell membran e receptor protein tyrosine kinase 7 (PTK 7), was used as the targeting
63 aptamer. CEM cells, which have a high expression of PTK 7, were chosen as target cancer cells, whereas Ramos cells with no PTK 7 on the membrane were used as control cancer cells. As s hown in Figure 2 5 A a large shift was observed for CEM cells treated with the SMNs, but no significant shift was observed for Ramos cells ( Figure 2 5 B ). Corresponding confocal fluorescence microscopy images were consistent with the flow cytometry results ( Figure 2 6 ): a strong green fluorescence was observed for CEM cells, but no distinct fluorescence was seen for Ramos cells. Compared with aptamer only, enhanced binding was observed for CEM cells treated with SMNs. As shown in Figure 2 5 C and 2 5 D a larg er shift was observed in the flow cytometry histograms, even though a smaller number of SMNs was used. This result could be attributed to multivalent interactions, i.e., the simultaneous binding event of multiple aptamers on SMNs to multiple PTK 7 receptor s on the cell membrane. Since the affinity of a ligand for its receptor is highly dependent on the three dimensional arrangement and valency of the targeting moieties, 189 equipping nanocarriers with multiple targeting ligands has become a popular strategy to enhance specific binding affinity to the target cells. The enhanced binding arises from the increased residence time of the ligands on the cell membrane, leading to greater incorporation. 190 Since no binding occurs between Ramos cells and the aptamers on the SMNs, neither shift nor enhancement was observed. Thus, the aptamer still maintains its binding capability towar d its target cells after being incorporated onto SMNs. We next investigated whether SMNs could be internalized into the target cancer cells. Since FITC is a pH sensitive dye and its fluorescence is greatly reduced in acidic environments, such as in lysosom es inside living cells, the aptamer was labeled with 3
64 TAMRA for confocal fluorescence microscopy. After incubating CEM cells with SMNs at 37 C for 2 h, a strong red fluorescence was observed by confocal fluorescence microscopy, but no distinct red fluor escence was seen for Ramos cells (data not shown). The sgc8 aptamer has previously been reported to enter cells through receptor mediated endocytosis, with the lysosome as its final destination inside living cells. 176 Therefore, a co localization study was ca rried out to determine if the SMNs also entered the lysosomes. While incubating the cells with free aptamer or SMNs, the lysosomes of CEM cells were labeled with a green dye known as lysosensor. From confocal fluorescence microscopy images ( Figure 2 7 ), a green fluorescence from the lysosensor was observed for the first channel, and a red fluorescence from the aptamer or the SMNs was seen for the second channel. In addition, the images overlapped well and produced a yellow fluorescence inside the CEM cells, indicating that both the aptamer and the SMNs could enter the cells and reside in their lysosomes. In addition, for CEM cells treated with SMNs, even though a similar green fluorescence was observed compared with cells treated with free aptamer, a much st ronger red fluorescence was seen with the concentration of SMNs at 1 nM and aptamer at 5 nM, indicating an improved uptake efficiency of SMNs to target cancer cells, which is most likely attributed to multivalent binding. This enhanced internalization and the low pH inside lysosomes, coupled with the instability of SMN pores in an acidic environment, should facilitate the release of the anticancer drug efficiently. Cell Viability and Proliferation Assay As demonstrated by a release kinetics study, SMNs loa ded with DOX show slow release of drug under physiological conditions, but rapid release in acidic environments, such as that found in lysosomes. Therefore, the high specificity of aptamers to their
65 target cells should allow efficient and selective cytotox icity to CEM cells. To prove this point, the in vitro cytotoxicities of SMNs only, DOX only and DOX SMNs to both CEM cells and Ramos cells were investigated. Since magnetite based magnetic nanoparticles ( M MNPs) possess excellent biocompatibility, a neglig ible effect of SMNs only on both cell lines was observed ( Figure 2 8 A ). For DOX only and DOX SMNs ( Figure 2 8 B and 2 8 C respectively ), they presented dose dependent cytotoxicity behavior to CEM cells as well as Ramos cells. Compared to the IC 50 of DOX onl y (0.39 50 SMNs, showing the enhanced killing efficacy of DOX SMNs. However, for Ramos cells, relatively weak drug potency of DOX SMNs was seen, which can be attributed to two possibl e reasons: (1) T here is minimal internalization of DOX SMNs to Ramos cells ; (2) DOX release from DOX SMNs to binding buffer (leaking) is small during the 2 h cell treatment. In addition, as sufficient to kill more than 80% of CEM cells. However, only around 20% of Ramos cells were killed when more than 80% of CEM cells were killed by DOX demonstrating the superior selectivity of DOX SMNs compared to conventional chemot herapy using DOX only. Summarizing the results from the cytotoxicity assay, one can easily draw the conclusion that enhanced killing efficacy and improved targeting specificity can be achieved by using SMNs. One factor that needs to be pointed out is that the cytotoxicity behavior difference between CEM cells and Ramos cells to DOX only resulted from their distinct susceptibilities to the drug. 168
66 Explore the Potential of Smart Multifunctional Nanostructure s for Magnetic Resonance Imaging Most biological samples exhibit virtually no magnetic background; therefore, magnetic nanoparticles ( MNPs ) have been used for highly sensitive measurements and superior contrast imaging in turbid or otherwise visually obscured samples without purification, allowing for ra pid assays. For M MNPs, the transverse (or spin spin) relaxation time (T 2 ) is typically used for biosensin g and MRI applications, since their transverse relaxivity is significantly larger than longitudinal relaxivity. Here, we investigated the potential of using SMNs for MRI. The imaging strategy is based on a self amplifying proximity assay using SMNs. 91 When many SMNs bind to their intended molecular target through the interaction between the receptors on target cancer cell membrane and the aptamers on SMNs, they act cooperatively to form microscale clusters. According to out er sphere theory, the relaxivity of a particle is directly proportional to its cross sectional area. 191 192 Consequently, when SMNs assemble into clusters in the presence of their target cancer cells, the effective cross sectional area increase exceeds the additive contribution from all SMNs on the cell membrane, resulting in a larger and more powerful magneti c dipole. This makes the aggregated SMNs more efficient in enhancing the net transverse relaxation of neighboring water protons, leading to a decreased T 2 Using a benchtop nuclear magnetic resonance relaxometer, we quantified the T 2 of surrounding water p rotons in SMNs only, SMNs incubated with CEM cells, and SMNs incubated with Ramos cells. Compared with SMNs only, SMNs treated with CEM cells showed a huge decrease in the T 2 of the surrounding water protons, while SMNs treated with Ramos cells showed no o bvious change ( Figure 2 9 A ). As an intended
67 molecular target for SMNs, PTK 7 receptor is highly expressed on CEM cells instead of Ramos cells. Therefore, effective cluster formation of SMNs and decreased T 2 of surrounding water protons are expected in the sample containing CEM cells, but not the Ramos cell sample. When a reversible bulk field dephasing effect caused by local field inhomogeneities is incorporated into T 2 its characteristic time is referred to as T 2 *. Compared to T 2 weighted MRI images, T 2 weighted MRI images have better contrast, since they exhibit considerably higher sensitivity to susceptibility differences. 193 Therefor e, T 2 weighted MRI images of SMNs only, as well as SMNs incubated with CEM cells and Ramos cells, were further obtained at four different SMN concentrations, and the results are shown in Figure 2 9 B Comparing the first column (SMNs only) and the third co lumn (SMNs treated with Ramos cells), similar darkness was seen since there is no specific interaction between Ramos cells and SMNs. However, much darker images were obtained for the second column (SMNs treated with CEM cells), consistent with the relaxati on measurements. Putting the T 2 relaxation measurements and T 2 weighted MRI images together, we demonstrated that the SMNs could specifically bind to their target cancer cells, effectively forming clustered structures, resulting in decreased T 2 and additi onal image contrast. Conclusions In summary, our pH sensitive SMNs demonstrated efficient release of anticancer drug at lysosome pH and great potential to be used as T 2 contrast agents. With the incorporation of targeting aptamers onto PHMNPs with acid lab ile pores, the resulting drug delivery platform offers several attractive features: (1) R apid release of toxic anticancer drug ; (2) E nhanced specific binding and cell uptake from the multivalent effect ; (3) D ecreased nonspecific killing of control cancer c ells ; (4) S imultaneous MRI
68 imaging. The improved specificity and internalization of our drug delivery platform and its rapid release of anticancer drug inside cancer cell lysosomes greatly facilitate the treatment of cancer with minimized systemic toxicity Together with passive targeting from the EPR effect, our drug delivery platform with active targeting from aptamers should have better therapeutic efficacy, especially for in vivo cancer treatment. Moreover, the great potential of using SMNs as T 2 contra st agents may enable real time monitoring of the cancer treatment progress.
69 Table 2 1. Detailed aptamer sequence information. Name Sequence NH 2 T 10 sgc8 FITC NH 2 TTT TTT TTT T AT CTA A CT GCT GCG CCG CCG GGA AAA TAC TGT ACG G T T AGA FITC NH 2 T 10 sgc8 TAMRA NH 2 TTT TTT TTT T AT CTA A CT GCT GCG CCG CCG GGA AAA TAC TGT ACG G T T AGA TAMRA NH 2 T 10 sgc8 NH 2 TTT TTT TTT T AT CTA A CT GCT GCG CCG CCG GGA AAA TAC TGT ACG G T T AGA Underscore indicates the full sequence of sgc8 aptamer.
70 Figure 2 1. Synthesis route for heterobifunctional PEG ligand. (DCC = N, N' dicyclohexylcarbodiimide DMF = N, N d imethylformamide )
71 Figure 2 2. Synthesis and characterization of SMNs. A ) Schematic illustrating the synthesis of SMNs. TEM images of B ) IMNPs; C ) HMNPs; and D ) PHMNPs. Inset of D ) shows the enlarged image of a representative PHMNP. The scale bars are 100 nm (10 nm for t he inset). E ) Dispersibility of PHMNPs and PPHMNPs in hexane and water. F ) Fluorescence intensity of PPHMNPs and SMNs (exc itation: 545 nm). (IMNP = iron magnetite core shell nanoparticle, HMNP = hollow magnetite nanoparticle, PHMNP = porous hollow magnetite nanoparticle, PPHMNP = PEGylated porous hollow magnetite nanoparticle, SMN = smart multifunctional nanostructure)
72 Figure 2 3 Mechanism o f SMNs for targeted cancer chemotherapy. Due to surface coating of aptam ers, DOX SMNs specifically enter target cancer cells through receptor mediated endocytosis and r eside in acidic lysosomes. This leads to SMN pore size enlargeme nt because of its acid sensitivity, facilitating t he release of entrapped DOX and the killing of target cancer cells. (SMN = smart multifunctional nanostructure, DOX SMN = doxorubicin loaded smart multifunctional nanostructure, DOX = doxorubicin ) Figure 2 4 Cumulative release of DOX from DOX SMNs at different pH values. DOX SMNs were dialyzed against PBS at pH = 5, pH = 6 and pH = 7.4 at 37 C The released DOX was measured by UV Vis absorption spectroscopy. Compared to physiological pH (pH = 7.4), fast er release was observed at lower pH values (pH = 5 and pH = 6). (DOX = doxorubicin, DOX SMN = doxorubicin loaded smart multifunctional nanostructure, PBS = phosphate buffered saline)
73 Figure 2 5. Flow cytometry histograms to monitor th e binding of SMNs to CEM cells (target cells) and Ramos cells (control cells). A) and B) SMNs specifically bound to CEM cells. C) and D) The specific binding betw een SMNs and CEM cells generated a stronger shift compared to the interaction between sgc8 and CEM cells. (SMN = s mart multifunctional nanostructure) Figure 2 6. Confocal fluorescence microscopy images to monitor the binding of SMNs to C EM cells and Ramos cells A strong green fluorescence was obtained for A) CEM cells, whereas no obvious fluorescence was observed for B) Ramos cells. (SMN = smart multifunctional nanostructure)
74 Figure 2 7. Co localization study of sgc8 aptamer and SMNs with lysosensor in CEM cells. A) sgc8 was well co localized with lysosensor. B) SMNs were also well co localized with lysosensor, but producing stronger red fluorescence signal. (SMN = smart multifunctional nanostructure) Figure 2 8. Cytotoxicity assay of CEM cells and Ramos cells treated with SMN only, DOX only, and DOX SMNs. A) SMNs only had negligible effect on CEM cells and Ra mos cells. Both B) DOX only and C) DOX SMNs presented dose dependent cytotoxicity behavior to CEM cells and Ramos cells. (SMN = smart multifunctional nanostructure, DOX = doxorubicin, DOX SMN = doxorubicin loaded smart multifunctional nanostructure)
75 Fi gure 2 9 Potential of using SMNs as T 2 contra st agents. A ) T 2 relaxation measurements and B ) T 2 weighted MRI images of SMNs, SMNs incubated with CEM cells, and SMNs incubated with Ramos cells.The concentration of SMNs in A ) is 10 g/mL, while their conce ntrations in B ) are l abeled on the right side of the figure. (SMN = smart multifunctional nanostructure)
76 CHAPTER 3 SEMIQU ANTIFICATION OF ATP IN LIVE CELLS USING NONSPECIFIC DESORPTION OF DNA FROM GRAPHENE OXIDE AS THE INTERNAL REFEREN CE Background Graphene oxide (GO) has been attracting considerable attention in recent years. Because of its rich chemical, optical, and mechanical properties, GO has been widely used for sensitive and selective detection of various biomolecules, includin g small molecules, 109 194 195 nucleic acids, 109 194 198 and proteins, 109 194 199 both in solution and in living cells. 114 200 These methods utilized two important properties of GO. First, GO can strongly adsorb single stacking interaction between the ring structures in the nucleobases and the hexagonal cells of G O. 110 Second, GO is a superquencher to a wide range of fluorophores via f rster resonance energy transfer (FRET) or nonradiative dipole dipole coupling. Therefore, ssDNA labeled with fluorophore can be adsorbed and quenched by GO. Upon addition of cDNA or a specific molecular target, ssDNA will be desorbed from GO and the fluorescence signal will be recovered. This is the basic mechanism for detecting biomolecules by the DNA aptamer/GO system. 109 Although this specific desorption of ssDNA from GO by target molecules has been well accepted, the nonspecific desorption of ssDNA from GO by nontarget proteins has not been reported. In one of our previous studies, 199 we disco vered a very interesting phenomenon. When the DNA aptamer/GO system was used to detect target insulin in solution, the nonspecific signals caused by other nontarget proteins were Cells Using Nonspecific Desorption of DNA from Graphene Oxide as the Internal Reference, Ana l. Chem. 84 8622
77 relatively high when the concentrations of these proteins excee paper, Lu et al. reported that when the concentrations of nontarget proteins were only 100 nM, the nonspecific signals were low. 109 We reasoned that nonspecific desorption of ssDNA from GO is concentration dependent; when the concentration of nontarget proteins exceeds a certain level, nonspecific desorption increases significantly, resulting in a strong false positive signal ( Figure 3 1 ). To confirm this hypothes is, we attempted to use different concentrations of nontarget proteins to test the nonspecific desorption of ssDNA from the DNA/GO system. Experimental Section DNAs ATP aptamer molecular beacon ( AAMB ) 5 TAMRA CAC CTG G GG GAG TAT TGC GGA GGA AGG TT ( PEG ) 6 CCA GGT G Dabcyl 3 ; Control molecular beacon ( CMB ) 5 Dabcyl GCG AG A CCG CCG CAT TTG ATC GAT A CT CGC TAMRA 3 ; ATP aptamer, 5 CAC CTG GGG GAG TAT TGC GGA GGA AGG TT TAMRA 3 ; control aptamer, 5 GCG AGA CCG CCG CAT TTG ATC GAT A TAMRA 3 ; internal refe rence ssDNA, 5 GCG AGA CCG CCG CAT TTG ATC GAT A FAM 3 The ATP aptamer is indicated by underlining, and the stem part for each MB is indicated in bold font. TAMRA = c arboxytetramethylrhodamine Dabcyl = 4 (dimethylaminoazo)benzene 4 carboxylic acid, FAM = f luorescein amidite Preparation of Graphene Oxide Generally, graphite powder (2 g, 325 mesh) was reacted with 12 mL of concentrated H 2 SO 4 3.0 g of K 2 S 2 O 8 and 3.0 g of P 2 O 5 at 80 C for 4.5 h wi th stirring. After cooling to room temperature it was di luted with 0.5 L of deionized water and left overnight. Then the mixture was centrifuged and washed to remove the residual acid
78 and dried overnight under ambient conditions. This preoxidized graphite was added into 120 mL of concentrated H 2 SO4 (0 C), and 15 g of KMnO 4 was gradually added with stirring in an ice bath. The reaction was permitted at 35 C for 2 h an d then diluted with 250 mL of deionized water in an ice bath to keep the temperature below 50 C. The mixture was stirred for 2 h, and another 0.7 L of deionized water was added. Afterward, 20 mL of 30% H 2 O 2 was added and a brilliant yellow color was observed along with bubbling. Washed by 1 L of 1:10 HCl a queous solution and by 1 L of deionized water, the resulting solid was dried in air and dilute d to make a GO dispersion (0.5% w/w). Finally, remaining impuritie s were removed by dialysis in deionized water for 1 week. As shown in Figure 3 2 the G O is a 2D sheet like material consisting of a densely packed sp 2 hybridized carbon atom network. They h there were no apparent aggregations. Fluorescence Response of ATP Aptamer Molecular Beacon and Control Molecular Beacon to ATP in Buffer All fluorescence measurements were performed using a Fluorolog spectrofluorome ter (Jobin Yvon Horiba). The MB samples were prepared in 10 mM Tris HCl buffer containing 6 mM MgCl 2 The AAMB and CMB concentrations were both 0.1 fluorescence spectra for all samples were measured at 20 C. Live Cell Imaging of ATP Using G raphene O xide to Deliver DNAs into Cells Generally, 200 nM DNAs were incubat Dulbecco s Modified Eagle Medium ( DMEM ) for 5 min. Then the solution was incubated with HeLa cells for 2 h. Cells were washed with phosphate buffered saline ( PBS ) and cultured for another 8 h in fresh DMEM followed by confo cal microscopy.
79 In Situ Semiquantification of ATP Generally, 200 nM MBs, or 200 nM internal reference, were incubated with 2.5 for 5 min, respectively. After combining, the mixture was incubated with HeLa cells for 2 h. Cells were washed with PBS and cultured for another 8 h in fresh DMEM Cells were then detected by confocal microscopy. For Ca 2+ or etoposide treatment, the further culture time was 2 h. Results and Disscussion Nonspecific Desorption of ssDNA from DNA/G raphene O xide Complex by Proteins A random 40 mer ssDNA library was generated with a FAM group label on the 5 end. As shown in Figure 3 3 upon excitation at 480 nm, the library (100 nM) gave a strong FAM emission at 520 nm. We prepared GO ( see the Experiment al Section and Figure 3 2 for details ), and addition of GO brought the fluorescence to the baseline level. However, when nontarget protein, bovine serum albumin (BSA), was added to the reaction solution, the fluorescence was restored, and a significant enhancement o ccurred when BSA reached 50 value. In addition, we tested the nonspecific desorption assay with a protein mixture, fetal bovine serum (FBS). As shown in Figure 3 4 the enhancement by FBS was comparable to that of BSA. A significant fluorescence signal was observed with FBS at These data indicated that ssDNAs can be nonspecifically desorbe d from GO by nontarget proteins, especially when they are at relatively high concentrations, resulting in high false positive signals. On the basis of these results, we concluded that this
80 nonspecific effect cannot be ignored when a DNA/GO system is used i n a matrix containing high concentrations of nontarget proteins, especially inside living cells. Consequently, it is necessary to consider how to reduce this false positive signal when a DNA/GO system is used for intracellular studies. To address this issu e, we designed a model study in which an ATP aptamer/GO system was used for in situ ATP detection. As the primary energy currency, ATP plays important roles in cell signaling and many cellular reactions. Because of its unique importance, many approaches ha ve been developed to detect ATP in solution, including high pressure liquid chromatography ( HPLC ) 201 aptamer based, 109 enzyme based, 202 203 and protein based methods. 204 For in situ detection, Wang et al. developed an ATP a ptamer/GO platform to detect ATP in living cells. 114 They cultured ATP aptamer/GO with cells in medium containing 10% FBS. When ATP aptamer/GO entered cells, cellular ATP bound to ATP aptamer s and released them from the surface of GO. Although their results showed a very clean background, the signal from the sample was rather weak, and therefore the difference between sample and control was small. Because ATP molecules in living cells are usua lly at very high concentrations, typically 1 10 mM, 205 assuming most of ATP aptamers were delivered into cells by GO and released from GO by intercellular ATP molecules, the recovered fluorescence signals should be strong It is questionable why the positive signal was so weak. From our previous nonspecific desorption data, we postulated that when aptamer/GO is cultured with cells in the presence of 10% FBS, some of the aptamers would be desorbed from GO by FBS and only th e remaining aptamers could be delivered into cells. As a result, the actual concentration of ATP aptamers on GO inside the cells would be decreased, resulting in a weak positive signal.
81 To confirm this, the ATP aptamer/GO system was cultured with HeLa cell s in two different media: one contained 10% FBS and the other was serum free. As shown in Figure 3 5 in the presence of FBS, the fluorescence signal was very weak, while in the absence of FBS, the fluorescence signal was strong. These data confirmed our p ostulate that some of the ATP aptamers were nonspecifically desorbed from the GO by FBS before entering the cells. This result also indicates that when using GO to deliver nucleic acids into cells, a serum free medium is preferable to a medium containing s erum. However, even though a serum free medium would allow most of the aptamer/GO to be delivered into the cells, the nonspecific release of ATP aptamers from GO by intracellular proteins would still occur, leading to false positive signals, as described a bove. To overcome this problem, we modified the ATP aptamer into a n AAMB Test the Feasibility of the Design in Buffer An aptamer molecular beacon, AMB, also called an aptamer switch probe 206 or an activatable aptamer probe, 207 is a newly developed molecular beacon which can specifically recognize target molecules, such as ATP, 206 proteins, 206 or even cells. 207 This type of MB is usually composed of three elements (in addition to the fluorophore and quencher on th e ends): an aptamer, a short DNA sequence complementary to part of the aptamer, and a linker (e.g., PEG) connecting the former two. Upon binding to their targets, AMBs undergo spontaneous structural reorganization, which opens the hairpin stem, leading to fluorescence recovery. We designed the AAMB based on one of our previous publications, 206 in which TAMRA was chosen as the fluorophore and Dabcyl was used as the quencher. The activity of AAMB to target ATP was first tested. As shown in Figure 3 6 A significant fluorescence enhancement was observed after the
82 AAMB was mixed with increasing concentrations of ATP in the reaction buffer. However, there was no fluorescen ce enhanc ement when CMB was used ( Figure 3 6 B ), indicating that CMB cannot bind to ATP. Before we used the AAMB to replace ATP aptamer in GO mediated cellular delivery, we first tested the nonspecific desorption caused by FBS on the ATP aptamer/GO system and the AA MB/GO system in solution. As shown in Figure 3 7 100 nM ATP aptamer produced much higher fluorescence than 100 nM AAMB when not adsorbed on GO. When GO was present, the fluorescence from both ATP aptamer and AAMB were severely quenched. However, when FBS (1.6 mg/mL) was added, ATP aptamer/GO produced a significant false positive signal while AAMB/GO did not. This experiment indicated that, although these DNAs can be nonspecifically released from GO by nontarget proteins, the self quenching ability of the A AMB results in a much smaller false positive signal. Intracellular ATP Detection Using A TP A ptamer M olecular B eacon /G raphene O xide Complex Next, we studied in situ ATP imaging by delivering AAMB or ATP aptamer with their corresponding control DNAs into cel ls by GO. Figure 3 8 A shows the confocal microscopy images of HeLa cells after incubation with AAMB/GO for 2 h, followed by further culture for another 8 h. Significant TAMRA fluorescence was observed. As a control, HeLa cells treated with CMB/GO under the same conditions showed only very weak fluorescence ( Figure 3 8 C ). By comparison, the background signal of the control aptamer was visible ( Figure 3 8 E ), and the difference in fluorescence intensity between the ATP aptamer and the control aptamer was small ( Figure 3 8 E and 3 8 F ). These data clearly demonstrated that the AAMB/GO system is much better than the ATP
83 aptamer/GO system for in situ ATP imaging because of : (1) M uch less false positive signal; (2) C lean background. The GO mediated delivery condition s were further optimized by treating AAMB with different amounts of GO. As shown in Figure 3 9 deliver 200 nM AAMB into HeLa cells. When the GO concentration was increased to 10 y (data not shown) due to the following two reasons: (1) T he concentration of AAMB complexed per GO particle decreases as the numbe r of GO particles increases; (2) N ot all GO particles enter the cell. Thus, the number of GO delivered AAMBs ac tually decreas es in the cell at The incubation time was also optimized. After incubation with the AAMB/GO complex for 0.5, 1, 2, or 3 h, respectively, HeLa cells were washed and further cultured for 8 h. The 2 h incubation time was found to be sufficient for AAMB/GO c omplex delivery into cells (data not shown). Moreover, for HeLa cells treated with AAMB/GO, more intense fluorescence was observed when the post treatment incubation period (after removal of excess AAMB/GO) was increased. As shown in Figure 3 10 the fluor escence signal from the 8 h incubation group is much stronger than that from the 2 h group. Intracellular ATP Semiquantification t hrough Raitometric Measurements Furthermore, on the basis of the finding of this nonspecific desorption, we designed an assay for in situ ATP semiquantification. As discussed above, the control aptamer/GO system gave a nonspecific signal inside living cells caused by nontarget cellular proteins ( Figure 3 8 E ). This signal should not be altered by different ATP concentrations insid e living cells. Therefore, the control aptamer can be used as an internal reference with AAMB as the probe for in situ ATP semiquantification. The
84 mechanism i s illustrated in Figure 3 11 An internal reference was prepared by replacing TAMRA on the con trol aptamer with a FAM group. Then internal reference/GO was mixed with AAMB/GO or CMB/GO for cellular delivery. As shown in Figure 3 12 internal reference/GO gave almost the same FAM signal in different groups of HeLa cells ( Figure 3 12 B and 3 12 E em = 52 0 nm), as we have assumed before. On the other hand, AAMB gave a much stronger signal compared to CMB ( Figure 3 12 A and 3 12 D em = 580 nm). These data demonstrated that, because of the similar cellular makeup, internal reference/GO would give stable refe rence signals at 520 nm and AAMB/GO would give signals specific to ATP at 580 nm. Based on this design, we then employed this AAMB internal reference/GO platform to detect different ATP levels in three groups of HeLa cells treated as follows: medium only, medium + 5 mM Ca 2+ medium + 0.1 mM etoposide. We used the advanced imaging probe design to measure intracellular ATP changes up on drug stimulation. Ca 2+ and etoposide are both known to induce intracellular ATP concentration enhancement. 208 209 As shown in Figure 3 13 the internal reference signals ( Figure 3 13 B 3 13 E and 3 13 H ) were very similar in the three groups of HeLa cells, but AAMB ( Figure 3 13 A 3 13 D and 3 13 G ) showed different fluorescence signals after Ca 2+ or etoposide treatment. The imaging data were collected in triplicate for each group, and software Image J was used to measure the signa l intensity. The averaged TAMRA signal intensity was divided by the averaged FAM signal intensity to get the quotient value for each group. For the control group, the quotient value was 1.419; for the Ca 2+ treated group, it was 2.313; and for the etoposide treated group, it was 2.753. As shown in Figure 3 14 semiquantification of
85 ATP concentration in situ was achieved. If the ATP concentration in the untreated HeLa cells is defined to be 100% (corresponding to an intensity ratio of 1.419), the ATP level in the cells treated with Ca 2+ increased to 163% (2.313/1.419), which is in good agreement with the results of others, 209 and cells treated with etoposide increased to 194% (2.753/1.419). These data clearly demonstrate that the AAMB internal reference/GO system is a viable tool to pe rform ATP semiquantification in living cells. Conclusions Our study offers several advantages. After the discovery of nonspecific DNA desorption from the DNA/GO system, we utilized the AAMB/GO, instead of ATP aptamer/GO, for cellular delivery and greatly r educed background for highly sensitive in situ ATP imaging. Moreover, using the nonspecific desorption as the internal reference, this platform can be used for a semiquantitative assay for intracellular ATP imaging, which is currently a challenging task. F inally, and more importantly, our aptamer MB/GO system has the potential to detect other biomolecules inside living cells, especially proteins having known aptamers. We plan to extend the excellent properties demonstrated in this platform to perform in sit u imaging of proteins in living cells. Taken together, we believe our study can serve as a basis for further design and optimization of GO mediated target detection and DNA delivery.
86 Figure 3 1. Nonspecific d esorption of ssDN A from GO by n ontarget p roteins Fluorescen tly labelled ssDNA molecules are adsorbed on GO to form a quenching platform. In the presence of nontarget pr oteins, some of these ssDNAs are release d nonspecifically from GO and g i ve false positive signals. (GO = graphene oxide) Figu re 3 2 TEM image of GO. S cale bar is 500 nm. (GO = graphene oxide)
87 Figure 3 3 Fluorescence spectra of FAM labeled ssDNA library o nly and FAM labeled ssDNA library/GO complex in the presence of different concentrations of BSA. Excitation: 480 nm and emission: 520nm ( GO = graphene oxide, BSA = bovine serum albumin ) Figure 3 4. Fluorescence intensity of FAM labeled ssDNA library/GO complex in the presence of different concentrations of FBS. Excitation: 480 nm, and emission: 520 nm. ( GO = graphene ox ide, FBS = fetal bovine serum )
88 Figure 3 5. Confocal microscopy images of HeLa cells treated with ATP aptamer/GO under two different conditions A) and B) C ulture mediu m contained 10% FBS ; C) and D) serum free medium (GO = graphene oxide FBS = fetal bo vine serum ) Figure 3 6. Fluorescence spectra of AAMB and CMB in the presence of ATP. A) AAMB treated with different concentrations of ATP. B) CMB treated with 1 mM ATP. Excitation: 565 nm, and emission: 580 nm. (AAMB = ATP aptamer molecular beacon, CMB = control molecular beacon)
89 Figure 3 7. Comparison of fluorescence intensities of AAMB and ATP aptamer under different conditions A) 100 nM AAM B or ATP aptamer in buffer ; B) AAMB or ATP aptamer treated wi ; C) AAMB/GO or ATP aptamer/GO treated with FBS (1.6 mg/mL). (AAMB = ATP aptamer molecular beacon, GO = graphene oxide, FBS = fetal bovine serum) Figure 3 8. Intracellular imaging of ATP. Confocal microscopy images of HeLa cells tr eated with A) and B) AA MB/GO ; C) and D) CMB/GO ; E) and F) control ssDNA/GO ; G) and H) ATP aptamer/GO (AAMB = ATP aptamer molecular beacon, GO = graphene oxide CMB = control molecular beacon )
90 Figure 3 9. Confocal fluorescence microscopy images of HeLa c ells treated with AAMB/GO at different concentrations of GO. A ) and B) 1.25; C ) and D) 2.5; E ) and AAMB was 200 nM in all cases. (AAMB = ATP aptamer molecular beacon, GO = graphene oxide)
91 Figure 3 10. Confocal fluorescence microscopy images of HeLa cells treated with AAMB/GO for 3 h and after further different incu bation ti mes. A ) and B) 2 h; C ) and D) 4 h; E ) and F) 8 h. (AAMB = ATP aptamer molecular beacon, GO = graphene oxide)
92 Figure 3 11. Advanced ATP i maging p r obe d esign A n AAMB is adsorbed on GO to form a double quenching platform. After the AAMB/GO com plex spontaneously enters cells, the AAMB is released and then opened by intracellular ATP. The resulting fluorescence em = 580 nm) is used to perform ATP live cell imaging. Moreover, in the presence of an internal reference, i.e., FAM em = 520 nm), which is released nonspecifically from GO when inside cells, this system can also be used for ATP semi quantification inside living cells. (AAMB = ATP aptamer molecular beacon, GO = graphene oxide) Figure 3 12 Confocal microscopy images of HeLa cells treated with advanced ATP imaging probes A), B), and C) AAMB internal reference/GO ; D), E), and F ) CMB internal reference/GO (AAMB = ATP aptamer molecular beacon, GO = graphene oxide, CMB = control molecular beacon)
93 Figure 3 13. ATP imaging with drug stimulation. Confocal microscopy images of HeLa cells incubated with AAMB internal reference/GO af ter treatmen t for 2 h with A), B), and C) medium only ; D), E), and F) medium + 5 mM Ca 2+ ; G), H), and I) ex em = 520 nm; for ex em = 580 nm. (AAMB = ATP aptamer molecular beacon, GO = graph ene oxide) Figure 3 14. Intrace llular imaging quantification. s emiquantification of ATP in living HeLa cells after treatment with 5 mM Ca 2+ or 0.1 mM etoposide, as described in Figure 3 13 caption.
94 CHAPTER 4 DNA MICELLE FLARES FOR INTRACELLULAR MRNA I MAGING AND GENE THERAPY Background The hybridization between a nucleic acid strand and its complementary sequence, one of the strongest and most specific molecular recognition events, 40 46 has greatly facilitated the development of disease diagnosis and gene therapy. For example, both linear 210 211 and hairpin 43 212 213 nucleic acid probes have been used to visualize and detect specific messenger RNAs (mRNAs) in living cells. Many mRNAs are disease related and can be used as specific biomarkers to assess the stage of these disease s, including cancer. Through molecular engineering, these probes can effectively translate an mRNA binding event into a fluorescence signal change without the need to remove unbound free probes. In addition, most human diseases, including cancer, could be treated with the introduction of genetic materials plasmid DNAs, 214 antisense oligonucleoti des, 153 215 small interfering RNAs, 216 small hair pin RNAs, 217 and microRNAs 218 into somatic tissues. These genetic materials can eit her enhance gene expression 219 or inhibit the production of deleterious proteins, thus making nucleic acid probes excellent candidates for gene therapy. 153 The advantages of nucleic acid probes lie in the simplicity of their synthesis, the suitability of their modifica tion, and the selectivity of their binding. 46 However, their potential has not been fully realized because of the following reasons. First, as negatively charged hydrophilic biomacromolecules, nuc leic acid probes cannot free ly traverse the cell membrane, 220 thus requiring additional Imaging and Gene Therapy, Angew. Chem. Int. Ed. 52 2012
95 instruments (such as microinjection or electroporation) or materials (such as transfection reagents, including cationic lipids/polymers and nanomaterials) for efficient cellular internalization. 215 Second, nucleic acid probes can be unstable even after successful cellular delivery because of endogenous nuclease digestion, 137 leading to high false positive signals or decreased therapeutic efficiency. Th ird, most applications for nucleic acid probes focus on either mRNA detection or gene therapy, while a better strategy to improve the patient outcome would be t he combination of mRNA imaging 213 221 222 and gene therapy 223 224 into one biomolecular method. Through mRNA imaging, real time spatiotemporal evaluation of nucleic acid probe delivery and target gene expression can be realized non invasively, providing useful information for assessing therapeutic efficiency, adjusting treatment methods, and refining probe design. 225 226 Even though nucleic acid functionalized gold nanoparticles (AuNPs) with efficient cellular uptake 227 an d enhanced enzymatic stability 228 have been developed to solve the first two challenges, they suffer non negligible cytotoxicity at relatively high concentrations as a result of AuNP inco rporation. 53 In addition, the preparation of these probes is very time consuming, requiring more than 24 h even after obtaining the AuNPs and nucleic acids. 215 Therefore, an ideal nucleic acid probe should be easy to synthesize, capable of self delivery, highly biocompatible, and sufficiently stable in a cellular environment, while at the same time, performing multiple functions in living cells. Herein, we present a sensitive and selective approach for combined mRNA detection and gene therap y using molecular beacon micelle flares (MBMFs). MBMFs are easily prepared by self as sembly of diacyllipid molecular beacon conjugates (L MBs), not requiring any biohazardous materials. Just like pyrotechnic flares that
96 produce brilliant light when activat ed, MBMFs undergo a significant burst of fluorescence enhancement upon target binding. This hybridization event subsequently induces gene silencing, leading to apoptosis of cancer cells. The advantages of MBMFs include easy probe synthesis, efficient cellu lar uptake, enhanced enzymatic stability, high signal to background (S/B) ratio, excellent target selectivity, and superior biocompatibility. In th is approach ( Figure 4 1 ), L MBs spontaneously self assemble into MBMFs with a diacyllipid core and an MB coro na in aqueous solutions owing to hydrophobic interactions. The MB part is a DNA sequence composed of a target recognition loop flanked by two short complementary stem sequences. The formation of the stem loop (hairpin) structure brings the quencher and flu orophore, which are located at the opposite ends of the MB, into close proximity, thus effectively quenching the fluorescence (OFF state). Upon hybridization to the target mRNA, the MB in MBMFs undergoes a conformational change that opens the hairpin struc ture, physically separating the fluorophore from the quencher and allowing fluorescence to be emitted upon excitation (ON state). In addition, the hybridization of MBMFs to the target mRNA can specifically inhibit gene expression through different mechanis ms, including translational arrest by steric hindrance of ribosomal activity and the induction of RNase H endonuclease activity, 229 leading to the suppression of cancer cell growth. Experimental Section Synthe sis of L ipid P hosphoramidite Synthe sis of compound 1 (see Figure 4 2 for structures): A solution of stearoyl chloride (5.00 g) in 50 mL of 1,2 dichloroethane was added dropwise to a solution of 1,3 diamino 2 dydroxypropane (0.73 g) and triethylamine (TEA) (2. 57 mL) in 100 mL of 1,2 dichloroethane. The reaction mixture was stirred under a blanket of Ar at room
97 temperature for 2 temperature. The solid product was filtered, washed with CH 2 Cl 2 CH 3 OH, 5% NaHCO 3 and CH 3 CH 2 OCH 2 CH 3 in order, and vacuum dried to yield compound 1 as a white solid. The product was identified by 1 H NMR according to dat a reported in the literature. 144 Synthesis of compound 2: N,N diisopropylethylamine ( DIPEA) (4.19 mL) was injected into a solution of compound 1 (3.00 g). The solution was cooled on an i ce bath under a blanket of Ar and then 2 cyanoethyl N,N diisopropylchlorophosphoramidite (2.15 mL) was added dropwise. The reaction mixture was stirred un der room temperature for 1 h and t temperature, washed with 5% NaHCO 3 and brine, Na 2 SO 4 dried, and vacuum concentrated. The product was precipitated as a white solid by adding concentrated solution into CH 3 CN. The product was ident ified by 1 H NMR and 31 P NMR according to data reported in the literature 144 Synthesis of Oligonucleotide P robes All oligonucleotide probes were synthesized on an automated ABI3400 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA, USA) through solid phase oligonucleotide synthesis starting from corresponding controlled pore glass (CPG). Lipid phosphoramidite and all others (e.g., pyrene phosphoramidite 144 ) were dissolved in CH 2 Cl 2 and CH 3 CN, respectively, an d then coupled onto oligonucleotide sequences. Oligonucleotide probes containing carboxytetramethylrhodamine (TAMRA) were deprotected in 3 mL TAMRA deprotection solution (methanol : tert butylamine : water = eprotected in 3 mL AMA solution oligonucleotide probes were precipitated by adding 250 L of 3 M NaCl and 6 mL of
98 cold CH 3 CH 2 OH. Then the precipitated oligonucleotide probes w ere collected by centrifugation at 4000 rpm for 30 min and dissolved in 400 L of triethylammonium acetate (TEAA) for further pur ification by reverse phase high pressure liquid chromatography ( RP HPLC) (ProStar, Varian, Walnut Creek, CA, USA). Oligonucleot ide probes with and without diacyllipid were purified using C4 and C18 column, respectively. The mobile phase was CH 3 CN TEAA solution. Finally, these oligonucleotide probes were quantified by measuring their absorbance at 260 nm using a Varian Cary 100 UV Vis spectrometer (Agilent Technologies, Santa Clara, CA, USA). Critical Micelle Concentration Critical micelle concentrations ( CMCs ) of MBMFs and phosphorothioate d MBMFs ( S MBMFs ) were determined by the distinguishable pyrene excimer fluorescence of the p yrene modified MBMFs (P MBMFs) and S MBMFs (PS MBMFs), respectively. Typically, P MBMFs and PS MBMFs with a concentration of 100 nM in phosphate buffered saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 2 H 2 O, 2.0 mM KH 2 PO 4 pH 7.4) containing 5 mM MgCl 2 were diluted in series of concentrations using the same buffer until the excimer fluorescence disapeared. The fluorescence spectra were measured by a fluorometer with the excitation wavelength of 350 nm. Agarose Gel Electrophoresis Each DNA sample (20 M, 10 L) was mixed with 4 L of glycerol and analyzed by 4% agarose gel at 90 V for about 45 min in 1x TBE buffer (89 mM tris(hydroxymethyl)aminomethane, 2 mM ethylenediamine tetraacetic acid (EDTA) and 89 mM boric acid, pH 8.0). The bands were stained by ethidium bromide (EB), visualized by UV illumination (312 nm), and photographed by a digital camera.
99 Dynamic Light Scattering and Zeta potential Measurements Dynamic light scattering ( DLS ) measurements were performed on a Microtrac Nanotrac operated in the molecular mode. Zeta potential measurements were performed on a Brookhaven ZetaPlus operated in the Doppler shift analysis mode with fixed s Fluorescence Measurements Unless otherwise stated, MBMFs were diluted to a concentration of 200 nM in PBS containing 5 mM MgCl 2 and treated with the desired amount of complementary, mismatched, or noncomplementary target. For the en zyme digest ion assay, the concentration of MBMFs and MBs was 1 M, while the concentration of DNase I was 1 U/mL. Cell Culture (DMEM) (American Type Culture Collection, Manassas, VA, USA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Carlsbad, CA) and 0.5 mg/mL penicillin streptomycin (PS) (Americ 2 HBE135 cells were maintained in RPMI 1640 medium (American Type Culture Collection, VA, USA), supplemented with 10% FBS and 0.5 mg/mL PS 2 HEK293 s minimal essential medium (EMEM) (American Type Culture Collection, VA, USA), supplemented with 10% FBS and 0.5 mg/mL PS in 5% CO 2 Cell Lysate Preparation HEK293 cells were plated in a 35 mm cell culture dish (Corning Incorporated, Corning, NY, USA) and grown around 80% confluency before the experiments. Cells
100 were washed twice with 1 mL PBS and then 0.5 mL of Tris HCl buffer without proteinase inhibitor was added to the cell culture dish. Finally, cells were scraped off the cell culture dish and ruptured by mechanical shearing using a douncer homogenizer. Confocal Laser Scanning Microscopy Experiments Cells were plated in a 35 mm confocal dish (coverglass bottom dish) (MatTek Corp., Ashland, MA, USA) and grown to around 80% confluency for 48 h be fore the experiment. Cells were washed twice with 1 mL PBS containing 5 mM MgCl 2 and then incubated with the proper probe at the desired concentration in PBS containing 5 mM MgCl 2 2 After incubation, cells were washed twice with 1 mL PBS containing 5 mM MgCl 2 dispersed in 1 mL PBS containing 5 mM MgCl 2 and then subjected to confocal imaging. For transferrin AlexaFluor 633 conjugate (Tf Alex633) (Invitrogen, Carlsbad, CA, USA) treatment, 2 L conjugates were added to th e diamidino 2 phenylindole (DAPI) (Invitrogen, Carlsbad, CA, USA) staining, 1 drop of DAPI was added to the washed cells after incubation, followed by another incubation of 10 15 min, and then the cells w ere subjected to confocal microscope imaging, using an Olympus FV500 IX81 confocal microscope (Olympus, Center Valley, PA, USA). Flow Cytometry Experiments Cells were plated in a 35 mm cell culture dish (Corning Incorporated, Corning, NY, USA) and gro wn to around 80% confluency for 48 h before the experiments. Cells were washed twice with 1 mL PBS containing 5 mM MgCl 2 and then incubated with the proper probe at the desired concentration in PBS containing 5 mM MgCl 2 for 2 h at 37 2 After incubation, cells were washed twice with 1 mL PBS containing 5 mM MgCl 2 2 dispersed in 500
101 L PBS and then subjected to flow cytometry analysis using a FACScan cytometer ( Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). Cytotoxicity Assay S MBMFs and noncomplementary S MBMFs was evaluated using the CellTiter 2 atmosphere. A sample of 3,000 A549 cells in 50 L fresh cell culture medium was seeded into each test well on a 96 well plate. After 12 h, medium was removed from the well after centrifugation at 1300 rpm for 4 min, and another 50 L of fresh medium was added. Then S MBMFs and noncompl ementary S MBMFs at the desired concentration in 50 L of fresh medium were added to the well. After 48 h treatment, medium was removed from the well, and another 100 L of fresh medium was added for an additional 24 h incubation. Finally, 20 L of CellTit er 96 reagent was added to the well, and the 96 well plate was subjected to absorption measurement at 490 nm using a VersaMax tunable microplate reader (Molecular Devices, Inc., Sunnyvale, CA, USA). Results and Discussion Synthe sis and Characterization of Molecular Beacon Micelle Flare s L MBs with the illustrated structure ( Figure 4 3A ) were prepared by directly coupling a diacyllipid phosphoramidite onto the 5 end of MBs using a fully automated DNA /RNA synthesizer, purified by RP HPLC (Figure 4 4A and 4 4B ) and characterized by ESI MSn ( Figure 4 4C ). The diacyllipid phosphoramidite was synthesized through a three step reaction according to our previously published procedure. 144 After purification, L MBs spontaneously form MBMFs in aqueous solution with a very low CMC (below 10 nM, Figure 4 5 ), indicating their excellent stability compared to polymer micelle systems. 230 The formation of MBMFs was further confirmed by both agarose gel
102 electrophoresis ( Figure 4 3B ) and DLS ( Figure 4 3C ). MBMFs migrated much slower than MBs without diacyllipid, suggesting the successful formation of larger micel le nanostructures. DLS measurements showed that MBMFs had a diameter of 17.1 nm. After adding a synthetic complementary target (cDNA), the diameter increased to 29.4 nm, while incubating MBMFs with a synthetic non complementary target (rDNA) resulted in negligible size increase. These results indicated that MBMFs maintaine d target recognition capability after the formation of a micellar structure and that the binding event did not disrupt the structural integrity of the micelle. Consistent results were also obtained from zeta mV were obtained for MBMFs only, MBMFs treated with cDNA, and MBMFs treated with rDNA, respectively ( Figure 4 3D ). Detailed sequence information for all the probes used can be found in Table 4 1 Evaluate the Performance of M olecular B eacon M icelle F lare s in Buffer System The performance of the MBMFs was first evaluated in a buffer system ( see the Experimental Section for detailed buffer components and concentration ). According to fluorescence spectroscopy results, the response of the MBMFs was spec ific to the target sequences added, with approximately tenfold signal increase for cDNA, which is much higher than previously mentioned AuNP nucleic acid conjugates, 53 215 231 but only minimal signal enhancement when rDNA was added ( Figure 4 6A ). In addition, MBMFs were able to differentiate between a perfec tly complementary target and mismatched targets ( Figure 4 6B ). The fluorescence signal of MBMFs exhibited dose dependent increases in response to cDNA concentrations from 0 to 1 Figure 4 6C ) with a wide dynamic range from 0 to 200 nM ( Figure 4 6C Inset ). These results demonstrate that MBMFs can effectively sig nal the presence of a target sequence with excellent
103 selectivity and sensitivity. To compare the stability of MBMFs and MBs towards enzymatic digestion, we incubated each with th e endonuclease DNase I (1 U/mL significantly greater than what would be found in the cellular environment) and monitored the fluorescence signal increase as a function of time. This experiment showed a much slower increase in fluorescence signal for MBMFs compared to MBs, indicating their enhanced stability owing to increased resis tance to enzymatic digestion ( Figure 4 6D ). A similar phenomenon was also observed fo r MBMFs and MBs in cell lysate Evaluate the Performance of M olecular B eacon M icelle F lare s in Living Cells After testing the feasibility of the MBMF approach with a synth etic target, the ability of MBMFs to permeate the cell membrane and detect target mRNA was further investigated. The loop region of L MBs was designed to be perfectly complementary to c raf 1 mRNA, a cancer biomarker and antisense therapeutic target of gre at significance in cancer diagnostics and theranostics. Non complementary MBMFs with a similar background signal, but little response to the target were used as controls. A549 cells were used to verify the ability of MBMFs to detect intracellular mRNA in cancer cells. These adenocarcinoma human alveolar basal epithelial cells come from cancerous lung tissue and have a high expression level of c raf 1 mRNA. 153 To obtain optimal results for intracellular detection of c raf 1 mRNA, we optimized both the probe concentration and the incubation time for all cell experiments. A549 cells cultured on coverglass bottom confocal dishes were incubated with 150, 300, and 600 nM MBMFs and then imaged under a confocal laser scanning microscope. Increasing fluorescence signal was observed for cells treated with increasing concentrations of MBMFs ( Figure 4 7 ). We noticed that the cells treated with 150 nM MBMFs did n ot
104 generate sufficient fluorescence signal to illuminate c raf 1 mRNA, while the cells treated with 600 nM MBMFs resulted in a poor signal to background (S/B) ratio. Therefore, the optimal probe concentration was 300 nM, which had the best combined fluores cence signal and S/B ratio. We also studied the influence of incubation time on the fluorescence signal by incubating A549 cells with 300 nM MBMFs for 2 or 4 h Because similar fluorescence intensity was observed for both times (data not sh own), 2 h was ch osen as the assay time for the remaining cell experiments. In addition, a co localization assay demonstrated that most of the fluorescence came from the cytoplasm, instead of the endosomes or lysosomes ( Figure 4 8 ), indicating that the signal was caused by the specific binding of MBMFs to c raf 1 mRNA. Under optimized conditions, confocal laser scanning microscopy results revealed that A549 cells treated with MBMFs ( Figure 4 9A ) displayed much more fluorescence than the population treated with a non complem entary control ( Figure 4 9B ) or MBs alone ( Figure 4 9C ), demonstrating the selectivity of the system and the need for the diacyllipid moiety for efficient self delivery. In comparison, a normal bronchial epithelial cell line HBE135 from healthy lung tissue which expresses significantly less c raf 1 mRNA, 232 d isplayed very low fluorescence We also used flow cytometry to collect fluorescence data for cells treated with MBMF s. Compared to confocal laser scanning microscopy, which allows imaging of only a small number of cells, flow cytometry can analyze thousands of cells per second, generating a quantifiable statistical average for a large population of cells, while eliminat ing cell to cell variation and experimental artifacts. The flow cytometry results were in excellent agreement with the confocal imaging: 2.61 and 1.08 times si gnal enhancement was observed for A549 and HBE135
105 cells, respectively, when incubated with compl ementary MBMFs ( Figure 4 9D ). Thus, while MBMFs work for synthetic target detection in our buffer system, these results also demonstrate that MBMFs are useful for intracellular mRNA detection in living cells. In addition, the MBMF approach can also differe ntiate cell lines with distinct mRNA expression levels, such as the cancerous and normal cells used here. M olecular B eacon M icelle F lare s for Gene Regulation Before they can be used for gene therapy, MBMFs must first hybridize with the mRNA. The probe then acts either by blocking translation of the targeted mRNA or by forming a DNA/RNA hybrid with the target mRNA, which can be d egraded by the enzyme RNase H. 229 Using these mechanisms, the MBMFs can be used for imaging guided gene therapy. 225 For this purpose, we used Raf genes, which code for serine/threonine specific protein kinases that play pivotal regulatory roles in the development an d maintenance of certain human malignancies. Substantial evidence supports the theory that antisense oligonucleotides targeted against c raf 1 kinase can specifically inhibit c raf 1 mRNA expression and tumor progression through the aforementioned mechanis ms when properly delivered. Therefore, we also tested the anti proliferative effect of MBMFs on cancer cells. Because gene therapy based on antisense oligonucleotides requires a long treatment period, S MBMFs were used to avoid any potential nuclease diges tion in living cells that would diminish the therapeutic efficiency. Experiments showed that this DNA backbone modification did not significantly affect the performance of S MBMFs compared to MBMFs because they showed similar low background fluorescence wi thout target or a non complementary target and maximal fluorescence with excess target ( Figure 4 10 ). In addition, the S MBM Fs had a comparable CMC (below 20 nM, Figure 4 11 ) as the MBMFs (below 10
106 nM, Figure 4 5 ). According to the cytotoxicity assay ( Figure 4 12 ), A549 cell growth was negligibly influenced by the treatment of cells with non complementary S MBMFs, indicating th e superior biocompatibility of our system compared to some metal nanoparticle systems. 53 However, their treatment with S MBMFs resulted in a marked inhibition of cell proliferation in a dose dependent manner, suggesting t hat MBMFs can be applied as an antisense therapy for cancer cells with high expression of c raf 1 mRNA. Conclusions In summary, we have presented a novel nanoprobe based on molecular assembly that can be used for combined mRNA detection and gene therapy. T he advantages of this approach include easy probe synthesis, efficient cellular uptake, enhanced enzymatic stability, high S/B ratio, excellent target selectivity, and superior biocompatibility. Instead of incorporating potentially biohazardous materials f or efficient nucleic acid probe delivery, simple modification of MBs with a diacyllipid group provided the resulting MBMFs with new properties that MBs do not have, such as self delivery and enhanced intracellular stability. In addition to their use in the context of mRNA imaging and gene therapy, MBMFs possess a hydrophobic cavity that could be filled with additional hydrophobic materials, such as magnetic contrast agents or anticancer drugs, showing great promise for constructing an all in one nucleic aci d probe capable of imaging, diagnosis, and therapy at the same time.
107 Table 4 1. Detailed sequence information for all oligonucleotide probes. Name Detailed sequence information MBMF Diacyllipid Dabcyl GCGAG TCC CGC CTG TGA CAT GCA TT CTCGC TAMRA 3 a Noncomplementary MBMF Diacyllipid Dabcyl GCGAG ACC GCC GCA TTT GAT CGA TA CTCGC TAMRA a P MBMF Diacyllipid Pyrene GCGAG TCC CGC CTG TGA CAT GCA TT CTCGC a S MBMF Diacyllipid Dabcyl *G*C*G*A*G *T*C*C *C*G*C *C*T*G *T*G*A *C*A*T *G*C*A *T* T *C*T*C*G*C TAMRA a, b Noncomplementary S MBMF Diacyllipid Dabcyl *G*C*G*A*G *A*C*C *G*C*C *G*C*A *T*T*T *G*A*T *C*G*A *T*A *C*T*C*G*C TAMRA a, b PS MBMF Diacyllipid Pyrene *G*C*G*A*G *T*C*C *C*G*C *C*T*G *T*G*A *C*A*T *G*C*A *T*T *C*T*C*G*C 3 a, b MB Dabcyl GCGAG TCC CGC CTG TGA CAT GCA TT CTCGC TAMRA a cDNA AAT GCA TGT CAC AGG CGG GA rDNA CAA CTG GGA GAA TGT AAC TG 1m DNA AAT GCA TGT A AC AGG CGG GA c 2m DNA AAT GCA A GT CAC A C G CGG GA c a Underline denotes base pairs in the stem. b Star denotes phosphorothioated bases. c Red denotes mismatched bases.
108 Figure 4 1 Schematic illustration of MBMFs for intracellular mRN A detection and gene therapy. L MBs self assemble into MBMFs and enter living cells. Before bindi ng their target mRNA, the fluorophore and the quencher of the MBMFs are in close proximity (OFF state). Hybridization between the loop region and the target mRNA separates the fluorophore and the quencher, producing a fluorescence signal (ON state) and DNA /RNA heteroduplex for RNase H action. Note: not all MBs are shown on the MBMF. (MBMF = molecular beacon micelle flare, L MB = diacyllipid molecular beacon conjugate MB = molecular beacon ) Figure 4 2 Detailed synthesis route for lipid phosphoramidite. (DIPEA = N, N d iisopropylethylamine )
109 Figure 4 3 Characterization of M BMFs. A ) Structure of MBMFs. Note : not all the bases are shown. B ) Agarose gel electrophoresis of DNA marker (lane 1 ); MBs (lane 2); MBs with cDNA (lane 3); MBMFs (lane 4); and MBMFs w ith cDNA (lane 5). C) DLS and D ) zeta potential measurements of MBMFs, MBMFs with cDNA, and MBMFs with rDNA ( MBMF = molecular beacon micelle flare, MB = molecular beacon, cDNA = synthetic complementary target, rDNA = synthetic non complementary target) Figure 4 4 Purification and characterization o f L MBs which can self assemble into MBMFs. HPLC profile of L MBs after A) synthesis and B) purification. C ) Mass spectrometry analysis of L MB s Found mass: 10905.9, expected mass: 10903.8 The sample was an alyzed via ESI MSn using negative mode. (L MB = diacyllipid molecular beacon conjugate MBMF = molecular beacon micelle flare)
110 Figure 4 5 CMC determination of MBMFs. Excitation wavelength was 350 nm. Because of the limited fluorescence of pyrene, 10 nM should be considered as the upper limit of CMC, rather than the actual value. (CMC = critical micelle concentration MBMF = molecular beacon micelle flare )
111 Figure 4 6. Perfo rmance evaluation of MBMFs in buffer system. A ) Fluorescence emission spectro scopy of MBM Fs treated with cDNA and rDNA. B ) Fluorescence kinetics spectroscopy of MBMFs treated with cDNA, 1m DNA, and 2m DNA C ) Response of MBMFs to cDNA with concentrations ranging from 0 to 1 r anging from 0 to 200 nM with an excellent linear relationship. D ) Fluorescence kinetics spectroscopy of MBMFs and MBs treated with DNase I. ( MBMF = molecular beacon micelle flare, cDNA = synthetic complementary target rDNA = synthetic non complementary ta rget 1m DNA = synthetic one base mismatched target, 2m DNA = synthetic two base mismatched target MB = molecular beacon )
112 Figure 4 7. Condition optimization of MBMFs in living cells. Confocal laser scanning microscopy images of A549 cells treated with 150, 300, and 600 nM A), C), and E) MBMFs and B), D), and F) n on complementary MBMFs. TAMRA fluorescence was pseudo colored red Scale bars: 20 m. (MBMF = molecular beacon micelle flare) Figure 4 8. Co localization assay of MBMFs with Tf Alex a 633 TAM RA fluorescence was pseudo colored red and Tf Alexa 633 fluorescence was pseudo colored green Scale bars: 20 m. ( MBMF = molecular beacon micelle flare, Tf Alexa 633 = transferrin AlexaFluor 633)
113 Figure 4 9. Investigation of MBMFs in living cells. Confo cal laser scanning microscopy images of A549 cells treated with 300 nM A) MBMFs; B ) non complementary MBM Fs, and C ) MBs. TAMRA fluorescence was pseudo colored red. Scale bars = 20 ) Flow cytometry results from A549 and HBE135 cells treated with 300 nM MBMFs and non complementary MBMFs. (MBM F = molecular beacon micelle flare, MB = molecular beacon)
114 Figure 4 10. Performance comparison between MBMFs and S MBMFs in buffer system. MBMFs and S MBMFs had similar fluorescence signal by themselves, after adding excess cDNA or rDNA, indicating phosp horothioate backbone ( MBMF = molecular beacon micelle flare, S MBMF = phosphorothioated MBMF cDNA = synthetic complementary target, rDNA = synthetic non complementary targe t ) Figure 4 11. CMC determination of S MBMFs. Excitation wavelength was 350 nm. Because of the limited fluorescence of pyrene, 20 nM should be considered as the upper limit of CMC, rather than the actual value. (CMC = critical micelle concentration, S M BMF = phosphorothioated molecular beacon micelle flare)
115 Figure 4 12. Cytotoxicity assay of A549 cells treated with S MBMFs and non complementary S MBMFs. (S MBMF = phosphorothioated molecular beacon micelle flare)
116 CHAPTER 5 ONE STEP FACILE SURFACE EN GINEERING OF HYDROPHOBIC NANOCRYSTALS WITH DESIGNER MOLECULAR RECOGNITION Background Nanometer scale crystallites, which possess unique size or shape dependent physical and chemical properties, have demonstrated substantial potential for biomedical appl ications, including molecular imaging, 233 234 disease diagnostics, 235 236 cancer therapy, 237 239 etc. For example, quant um dots (QDs) have been used to sensitize photodynamic therapy (PDT) agents, leading to a novel class of PDT sensitizers with tunable optical properties for treating both shallow and deep seated tumors. 240 However, high quality nanocrystals are typically synthesized in organic solvents at e levated temperatures, resulting in bioincompatible nanocrystals coated with hydrophobic surfactant stabilizers (such as oleylamine, oleic acid, and any other hydrocarbon chain containing ligands). 72 241 To address this issue, two major strategies have been devised for hydrophobic nanocrystal surface engineering to generate soluble and stable nanocrystals in aqueo us solutions: (1) L igand exchange with thiol phosphine or do pamine containing molecules; (2) L igand encapsulation by a layer of amphiphilic polymers or silica shell. 242 244 Unfortunately, despite recent advances, these surface engineering approaches often fail to produce individually dispersed nanocrystals in various biological fluids. In addition, complicated procedures and intensive energy input (e.g., heat and sonication) are generally required. 83 Most T. et al. One Step Facile Surface Engineering of Hydrophobic Nanocrystals with Designer Molecular Recogition. J. Am. Chem. Soc. 134 13164 13167
117 importantly, additional steps are needed to incorporate biological moieties for specific molecular recognition, making the entire process time and labor consuming. Here, we demonstrate a novel one step method for hydrophobic n anocrystal surface engineering to produce different types of water soluble nanocrystals with tunable molecular recognition, using chimeric DNA molecules containing both hydrophobic (diacyllipid) and hydrophilic (oligonucleotide) parts. Through hydrophobic interactions, whereby relatively apolar molecules aggregate in aqueous solutions, 245 these chimeric DNA molecules spontaneously intercalate in the surfactant layer of hydr ophobic nanocrystals using their hydrophobic parts, encapsulating an individual nanocrystal inside the diacyllipid core and leaving an oligonuc leotide corona outside (Figure 5 1 ). The resulting water soluble nanocrystals have a relatively narrow hydrodynam ic size distribution and long term stability in various biological media. In addition, since DNA can specifically recognize their targets by either Watson Crick base pairing or by folding into distinct tertiary structures, these functionalized nanocrystals possess excellent selectivity to a variety of biomolecular targets, varying from nucleic acids to cancer cells. Experiment al Section Synthesis and Characterization of Hydrophobic Nanocrystals Synthesis of hydrophobic nanocrystals Synthesis of 13 nm Fe Fe 3 O 4 core shell magnetic nanoparticles (CSNPs). The 13 nm Fe Fe 3 O 4 CSNPs were synthesized using a modified protocol. 183 246 Oleylamine (0.3 mL, 0.9 mmol) was mixed with 20 mL 1 octadecene. The mixture was Ar at that temperature for 30 min to remove moisture and oxygen. Then, the temperature of the mixture was further increased to 180
118 5 was quickly injected into the mixture with vigorous stirring under Ar temperature before being exposed to air. After discarding the supernatant, the Fe Fe 3 O 4 CSNPs coated on the magnetic stirring bar were transferred to a centr ifuge vial and washed with hexane in the presence of oleylamine. After addition of isopropanol, the precipitated Fe Fe 3 O 4 CSNPs were collected by centrifugation at 12,000 rpm for 20 min. After repeating this procedure 3 times, the purified Fe Fe 3 O 4 CSNPs w ere dispersed in Since the amorphous Fe 3 O 4 shell of the as synthesized nanoparticles is not stable in dispersion state, it was further oxidized to produce a stable crystalline Fe 3 O 4 shell usin g an oxygen transferring reagent. Five mg (CH 3 ) 3 NO was mixed with 20 mL 1 Ar at that temperature for 30 min to remove moisture and oxygen. Then, 80 mg as synthesized nanoparticles dispersed in 2 mL hexane were quickly injected into the mixture wi th vigorous stirring under Ar 30 min, and then cooled to room temperature before being exposed to air. After adding isopropanol, the stabilized Fe Fe 3 O 4 CSNPs as a black precipitate were collected by centrifuge at 12,000 rpm for 20 min. The collected Fe Fe 3 O 4 CSNPs were then dispersed in hexane and precipita ted by adding isopropanol. After repeating this procedure 3 times, the purified Fe Fe 3 O 4 CSNPs were dispersed in hexane with 40 L
119 Synthesis of 7 nm Fe 3 O 4 nanoparticles (NPs). The 7 nm Fe 3 O 4 NPs were synthesized using a modified protocol. 247 Fe(acac) 3 (0.71 g 2 mmol), 2 mL oleic acid (~6 mmol) and 2 mL oleylamine (~4 mmol) were mixed with 20 mL phenyl ether under Ar with vigorous stirring. Then 2.58 g 1,2 hexadecanediol (10 mmol) was added, and to room temperature before being exposed to air. After adding ethanol, the Fe 3 O 4 NPs as a black precipitate were collected by centrifuging at 12,000 rpm for 20 min. The collected Fe 3 O 4 NPs were then dispersed in hexane and precipitated by adding ethanol. After repeating this procedure 3 times, the purified Fe 3 O 4 NPs were dispersed in hexane with 40 L Synthesis of 15 nm Fe 3 O 4 nanoparticles (NPs). The 15 nm Fe 3 O 4 NPs were provided as a generous gift from Dr. Tie Wang in the laboratory of Dr. Y. Charles Cao, Department of Chemistry, University of Florida. Synthesis of 13 nm Au nanoparticles (Au NPs). The 13 nm Au NPs were synthesized using a modified protocol. 248 HAuCl 4 2 O (0.196 g, 0.5 mmol) and 1.5 ml oleylamine (~3 mmol) were mixed with 10 mL phenyl ether. After the addition of 0.516 g 1,2 hexadecanediol (2 mmol), the temperature of the mixture was slowly increased to Ar The mixture was cooled to room temperature before being exposed to air. After adding ethanol, the precipitated Au NPs were collected by centrifugation at 12,000 rpm for 20 min. The collected Au NPs were then dispersed in hexane and precipitated by adding ethanol. After repeating this procedure 3 times, the purified Au NPs were dispersed in hexane with 40 L oleylamine or future use.
120 Synthesis of 6 nm CdS/ZnS quantum dots (QDs). The 6 nm CdS/ZnS QDs were synthesized using a modified protocol. 249 Cadmium myristate (1.0 mmol) and sulfur (0.5 mmol) were mixed with 50 g of 1 octadecene. The mixture was degassed at room temperature and Ar The nanocrystal growth was monitored using UV Vis spectroscopy. When the nanocrystal size reached 3.1 nm in diameter, the mixture was cooled to room temperature. After adding acetone, the precipitated CdS nanoparticles ( NPs ) were collected by centrifuging at 14,000 rpm for 30 min and redispersed in toluene. ZnS shells were grown onto the as synthesized CdS NPs in a mixture of 1 octadecence and oleylamine (volume ratio of 3:1). Zinc stearate in 1 octadecene (40 mM) and oleyla mine (40 mM) in 1 octadecene were injected alternatively with a growth time of 10 min after each injection. As soon as the desired shell thickness was achieved, as calculated by the method of Mews, 250 the mixture was cooled to room temperature. After adding acetone, the precipitated CdS/ZnS QDs were collected by centrifugation at 14,000 rpm for 30 min. The collected CdS/ZnS QDs were then dispersed in hexane and precipitated by adding ethanol. After rep eating this procedure 3 times, the purified CdS/ZnS QDs were dispersed in hexane with 40 L Synthesis of Fe Pt nanorods (NRs). Fe Pt NRs were synthesized using a modified protocol. 251 Pt(acac) 2 (49.2 mg, 0.125 mmol), 50 mg 1,2 hexadecanediol (0.2 mmol), and 6 mL oleylamine (~12 mmol) were mixed with 2 mL octyl ether. The mixture was stirred vigorously under Ar Then the temperature of the mixture was inc reased to Fe(CO) 5 (0.025 mmol) was quickly injected into the mixture. After another 20 min, 3 ml
121 oleylamine (~6 mmol) was quickly injected into the mixture. The temperature of the was cooled to room temperature before being exposed to air. After adding ethanol, the precipitated Fe Pt NRs were collected by centrifuging at 6000 rpm for 20 min. T he collected Fe Pt NRs were then dispersed in hexane and precipitated by adding ethanol. After repeating this procedure 3 times, the purified Fe Pt NRs were dispersed in hexane Synthesis of Au Fe 3 O 4 dimer nanoparticles (DNPs). Au Fe 3 O 4 DNPs were synthesized using a modified protocol. 252 HAuCl 4 2 O (0.1 g, 0.25 mmol) an d 10 mL oleylamine (~20 mmol) were mixed with 10 mL tetralin at room temperature and initially stirred for 10 min. Tetra n butylammonium bromide (TBAB) (1 mmol) and 1 mL oleylamine (~2 mmol) were mixed with 1 mL tetralin by sonication and quickly injected into the above solution. The mixture was further stirred at room temperature for 1 h. After adding ethanol, the precipitated Au nanoparticle ( NP ) seeds were collected by centrifuging at 14,000 rpm for 20 min. The collected Au NP seeds were then dispersed i n hexane and precipitated by adding ethanol. After repeating this procedure 3 times, the purified Au NP seeds were dispersed in hexane with 40 L oleylamine and stored at Ten mg as synthesized Au NP seeds (4 nmol) dissolved in 1 mL h exane, 0.5 mL oleylamine (~1 mmol) and 1 mL oleic acid (~3 mmol) were mixed with 10 mL 1 temperature for 20 min under gentle Ar flow to remove hexane. Then 50 L Fe(CO) 5 wa s quickly injected into the mixture under Ar The temperature of the mixture was
122 was cooled to room temperature before being exposed to air. After adding isopropanol, the p recipitated Au Fe 3 O 4 DNPs were collected by centrifuging at 12,000 rpm for 20 min. The collected Au Fe 3 O 4 DNPs were then dispersed in hexane and precipitated by adding isopropanol. After repeating this procedure 3 times, the purified Au Fe 3 O 4 DNPs were dis Transm ission electron microscopy images of hydrophobic nanocrystals Transmission electron microscopy ( TEM ) images were obtained on a Hitachi H 7000 transmission electron microscope at 100 kV. Five L samples of hydrophobic nanocrystals in their hydrophobic solvents were dropped onto 3 mm copper grids covered with a continuous carbon film. The samples were air dried at room temperature. Synthesis and Characterizati on of Amphiphilic Oligonucleotides All amphiphilic oligonucleotide sequen ces were synthesized on the ABI 3400 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA, USA) on a 1.0 micromolar scale using the corresponding controlled pore glass (CPG). As sy nthesized diac yllipid phosphoramidite 144 was dissolved in dichloromethane to a concentration of 0.1 M and then coupled using the DNA /RNA synthesizer. Detailed sequence information for all the amphiphilic oligonuc leotides is provided in Table 5 1 After the synthesis, the amphiphilic oligonucleotides were cleaved and deprotected from the CPG in 3 mL AMA All deprotected sequences were precipitated by adding 250 L 3 M NaCl and 6 mL cold ethanol and collected by centrifuging at 4,000 rpm for 30 min. After dissolving in 200 L triethylammonium acetate (TEAA), amphiphilic oligonucleotide sequences were purified by reverse phase high pressure liquid chromatograp hy (RP HPLC) (ProStar, Varian,
123 Walnut Creek, CA, USA) using a C4 column (BioBasic 4, 200 mm x 4.6 mm, Thermo Scientific, USA) with 0.1 M TEAA and acetonitrile as the eluent. Finally, the purified amphiphilic oligonucleotide sequences were quantified by mea suring their absorbances at 260 nm on a Cary Bio 100 UV spectrometer (Varian, Palo Alto, CA, USA). Synthesis and Characterization of Functionalized Hydrophobic Nanocrystals Synthesis of functionalized nanocrystals As prepared hydrophobic nanocrystals in th eir hydrophobic solvents (i.e., hexane or toluene) were precipitated by adding a polar solvent (i.e., isopropanol or acetone), transferred to t etrahydrofuran ( THF ) and adjusted to a concentration of 500 g/mL. To a 250 L aliquot of nanocrystal solution i n THF were added 25 L of amphiphilic oligonucleotide solutions in water with desired concentrations: 50, 250, 500, and 1000 M. The reaction was conducted at room temperature while shaking at 500 rpm in a 0.5 mL Eppendorf tube for 2 h. After the reaction, modified nanocrystals were first collected by centrifuging the reaction mixture at 14000 rpm for 10 min, then washed 2 times with 200 L water to remove excess amphiphilic DNA, and finally re dispersed in either water or PBS for future use. Characterizati on of functionalized nanocrystals Determination of the number of ligand per functionalized hydrophobic nanocrystal. To determine the number of ligand per functionalized hydrophobic nanocrystal, fluorescently labeled chimeric DNA molecules were used and the ir caliberation curves were first obtained by measuring their fluorescence intensity as a function of concentration. Then the fluorescence intensity of functionalized nanocrystals was measured and the ligand concentration of this sample was calculated out using previously obtained caliberation curve. Finally, the number of ligand per functionalized
124 nanocrystal was calculated out by dividing the molar concentration of the ligand with the molar concentration of the nanocrystal. All the fluorescence measuremen ts were done on a fluorometer using a 100 L cuvette. T ransmission electron microscopy images of functionalized hydrophobic nanocrystals. TEM images of functionalized hydrophobic nanocrystals were obtained on the same instrument and using the same experime ntal condit ions as specified before Since carbon film is hydrophobic and can cause false negative nanocrystal aggregation, copper grids were first glow discharged to become hydrophilic. Then 5 L functionalized hydrophobic nanocrystals in water were dropp ed onto the treated copper grid, and the sample was air dried at room temperature. UV Vis spectra of functionalized hydrophobic nanocrystals. UV Vis spectra of unmodified nanocrystals in their hydrophobic solvents and modified nanocrystals in water were re corded on a Cary Bio 100 UV spectrome ter using a 200 L quartz cuvette. Fo urier transform infrared spectra of functionalized hydrophobic nanocrystals. Fourier transform infrared ( FT IR ) spectra of vacuum dried unmodified and modified nanocrystals were reco rded on a Nicolet 6700 FT IR spectrometer (Thermo Scientific, West Palm Beach, FL, USA) in KBr pellets. Dynamic Light Scattering measurement of functionalized hydrophobic nanocrystals. Dynamic light scattering ( DLS ) data for functionalized hydrophobic nano crystals in water were obtained on a ZetaPALS DLS detector (Brookhaven
125 Binding of Functionalized Hydrophobic Nanocrystals with Nucleic Acid T argets Fe Fe 3 O 4 CSNP s were functionalized with amphiphilic olig onucleotide (lipid 20 and lipid T 20), as described before Streptavidin coated silica microspheres (SiMSs) (1 m) were modified with complementary DNA (C 20). With a concentration of 10 mg/mL, the binding capacity of SiMSs to biotinylated molecules was 0.36 M. Fifty L as purchased silica microspheres in buffer (100 mM borate, 10 Mm e thylenediaminetetraacetic acid ( EDTA ) 1% bovine serum albumin ( BSA ) 0.1% NaN 3 0.05% Tween 20, pH = 8.5) were washed three times with hybridization buffer ( 2 0 mM Tris HCl, 50 mM NaCl, 5 mM Mg Cl 2 ) and redispersed in 50 L hybridization buffer. Then, 1 L C 20 with a concentration of 500 M was added t o the washed SiMSs and the mixture was incubated while shaking f or 1 h at room temperature. After the reaction, C 20 coated SiMSs were washed 3 times with hybridization buffer and redispersed in hybridization buffer. Following this, 50 L functionalized Fe Fe 3 O 4 CSNPs with a concentration of 500 g/mL were added to the washed DNA microspheres, and the mixture was incubated while shaking for 4 h at room temperature to ensure maximal hybrid ization. After the reaction, Fe Fe 3 O 4 CSNP coated silica SiMSs were collected by centrifuging at a very low speed (i.e., 1500 rpm) for 3 min, washed 3 times with hybridization buffer and redispersed in 100 L hybridization buffer. Scanning electron microcopy (SEM) images were obtained on an FE S 4000 scanning electron microscope (Hitachi, Tokyo). A 5.0 L sample in hybridization buffe r was spread on a piece of microglass slide mounted on a specimen stub using double sided adhesive tape. The sample was then dried overnight in a desiccator, sputter coated with an ultrathin layer of gold, and then subjected to SEM imaging.
126 Binding of Functionalized Hydrophobic Nanocrystals with Cancer Cell Target The Fe Fe 3 O 4 CSNP s were functionalized with amphiphilic apt amer as described before To demonstrate the specific interaction between amphiphilic aptamer functionalized Fe Fe 3 O 4 CSNP s and canc er cells, fluorescence measurements were obtained on a FACScan cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA) using a 488 nm laser as the excitation source. CEM or Ramos cells in culture medium were washed three times with washing buffer and then redispersed in binding buffer at a concentration of 10 6 cells/mL. To 200 L CEM or Ramos cells in binding buffer, either aptamer alone or amphiphilic aptamer functionalized Fe Fe 3 O 4 CSNP s with desired concentrations were added, and the mixt ure was incubated on ice for 30 min. The cells were washed three times with washing buffer, redispersed in binding buffer, and subjected to flow cytometry analysis by counting 10,000 events. Magnetic Resonance Imaging T 2 weighted magnetic resonance imaging (MRI) was acquired on a 11 T/470 MHz MRI spectrometer (Bruker Optics, Billerica, MA). Amphiphilic aptamer modified Fe Fe 3 O 4 CS NPs were incubated with binding buffer only, CEM cells, or Ramos cells in binding buffer on ice for 30 min in 500 L binding buff er in 1.5 m L Eppendorf tubes. The final concentration for modified Fe Fe 3 O 4 CSNP s concentration of cells was 10 6 cells/mL. After incubation, the samples in Eppendorf tubes were vortexed, fixed on a homemade foam sample holder in a 1x3 array, and then put in the coil. T 2 weighted MRI images were acquired with a spin echo sequence.
127 Results and Discussion Development and Characterization of the Surface Engineering Approach The chimeric DNA molecules were synthesized by efficiently incorporating a diacyllipid at the 5 end of oligonucleotides through solid phase DNA synthesis on a fully automated DNA/RNA synthesizer, according to our previously reported procedure. 137 144 To t est the feasibility of this facile surface engineering method, oleylamine coated Fe Fe 3 O 4 CSNPs (13 nm) were first used. The as prepared Fe Fe 3 O 4 CSNPs were sp herical and fair ly monodisperse, as shown in the TEM image ( Figure 5 2A ). For functionalization, Fe Fe 3 O 4 CSNPs in THF a water miscible organic solvent, were mixed with chimeric DNA molecules in water. The reaction was conducted under an ambient atmospher e while shaking. After excess chimeric DNA molecules were removed by washing, the resulting Fe Fe 3 O 4 CSNPs were readily dispersed in water ( Figure 5 2C ) with negligible aggregation ( Figure 5 2B ). DLS and zeta potential measurements indicated that the as pr epared Fe Fe 3 O 4 CSNPs in hexane had a diameter of 16.2 nm ( Figure 5 3 ) and a zeta potential of 6.06 mV, respectively. Yet, the modified Fe Fe 3 O 4 CSNPs in water had a diameter of 27.2 nm (Figure 5 3) and a zeta potential o Table 5 2 ), respectively. These results suggested that hydrophobic Fe Fe 3 O 4 CSNPs were stabilized by chimeric DNA molecules in water and formed uniformly distributed nanoparticles. The maximal concentration that functionalized Fe Fe 3 O 4 CSNPs c an reach in aqueous solution is more than 5 mg/mL, which is sufficient for most of their biomedical applications. In addition, the engineered Fe Fe 3 O 4 CSNPs preserved the magnetic properties of the original nanocrystals ( Figure 5 2D ). Both UV visible and F T IR spectroscopies verified the presence of chimeric DNA molecules on the surfaces of functionalized Fe Fe 3 O 4 CSNPs ( Figure 5 4
128 and 5 5 ): the characteristic UV absorption peak around 260 nm and vibrational band between 750 and 1750 cm 1 belonging to DNA w ere easily identified. The surface density of chimeric DNA molecules on the modified Fe Fe 3 O 4 CSNPs was estimated by fluorescence measurements ( see the Experimental Section for detailed procedures ). With the newly developed surface engineering method, the number of chimeric DNA molecules per functionalized nanoparticle increased with ligand concentration and reached a plateau at the saturation concentration ( Figure 5 6 ). Using fluorescently labeled lipid T20, the saturation concentration for 0.5 mg/mL Fe Fe 3 O 4 modified nanoparticle at that concentration. In order to obtain functionalized nanoparticles with the desired solubility in aqueous environments, enough chimeric DNA molecules were n eeded; Fe Fe 3 O 4 CSNPs modified with adequa te chimeric DNA molecules ( 10 M) are soluble upon adding water, whereas the ones engineered with insufficient ligands ( 5 M) need vigorous vortexing or even sonication ( Figure 5 7 ). In addition, chimeric DNA mol ecules with varying lengths (from 5 to 60 nt) and distinct sequence information were all found to generate functionalized nanoparticles with excellent water dispersity ( Figure 5 8 ). Moreover, the hydrodynamic diameter and zeta potential of modified nanopar ticles increased and decreased, respectively, with the length of chim eric DNA molecules (Table 5 2 ). Since this novel surface engineering approach does not rely on the properties of the nanocrystal core or the reactivity of the nanocrystal surface, it coul d be generalized for many hydrophobic nanocrystals with variable size, composition, and morphology. To verify this point, chimeric DNA molecules were used to engineer Fe 3 O 4 NPs with two
129 different sizes (7 and 15 nm diameter). Water soluble Fe 3 O 4 NPs with m inimal aggregation were obtained under both circumstances ( Figure 5 9 A and 5 9 B ). In addition, this method worked equally well for nanocrystals with other compositions (CdS/ZnS QDs and Au NPs) an d morphologies (Fe Pt NRs and Au Fe 3 O 4 DNPs) ( Figure 5 9 C 5 9 D 5 9 E and 5 9 F ). A detailed synthesis procedure for these hydrophobic nan ocrystals can be found in the Experimental Section Therefore, the surface engineering method demonstrated here is independent of nanocrystal size, composition, and morphology. In addition, it is highly efficient with only a few empty micelles remaining ( Figure 5 10 ). In order to determine their utility in biomedical applications, the stability of engineered nanocrystals was evaluated in various solution environments. No obvious ag gregation was observed in water, phosphate buffered saline (PBS) or cell culture medium, even after more than 6 months. Tunable Molecular Recongition of Engineered nanocrystals After confirming the stability of functionalized nanocrystals in biological sy stems, we systematically investigated their ability to recognize desired molecular targets. First of all, the hybridization between modified nanocrystals and their complementary DNA (cDNA) was studied ( Figure 5 11 A ). Fluorescein isothiocyanate (FITC) label ed lipid 20 and FITC labeled lipid T20 were used as target and control chimeric DNA molecules, respectively. The cDNA here was perfectly complementary to lipid 20. The cDNA conjugated silica microspheres (cDNA SiMSs) were prepared by immobilizing biotinyla ted cDNA onto strepavidin coated SiMSs (SA SiMSs) and then incubating with engineered Fe Fe 3 O 4 CSNPs under an ambient atmosphe re in hybridization buffer while shaking for 4 h. For cDNA SiMSs treated with lipid 20 functionalized Fe Fe 3 O 4
130 CSNPs, a strong gre en fluorescence signal was observed by confocal laser scanning microscopy indicating a high degree of hybridization, while no fluorescence was seen for cDNA SiMSs mixed with lipid T20 modified Fe Fe 3 O 4 CSNPs, suggesting no hybridization ( Figure 5 11 B ). In addition, coating of lipid 20 functionalized Fe Fe 3 O 4 CSNPs (instead of lipid T20 modified Fe Fe 3 O 4 CSNPs) onto cDNA SiMSs was viewed b y SEM ( Figure 5 11 C ), further showing the selective binding of engineered Fe Fe 3 O 4 CSNPs to their nucleic acid target. I n addition, SEM images also illustrated that the hybridization event did not disrupt the structure integrity of engineered Fe Fe 3 O 4 CSNPs. Next, we tested the binding between modified nanocrystals and their target cancer cells, which is of great significan ce to their use in early cancer diagnosis and efficient cancer therapy. To accomplish this, Fe Fe 3 O 4 CSNPs were functionalized with an FITC labeled chimeric aptamer. Aptamers, generated from a process known as SELEX (Systematic Evolution of Ligands by EXpo nential enrichment), are single stranded oligonucleotides which can bind to their targets with high affinity and excellent selectivity by folding into distinct secondary or tertiary structures. 29 S gc8 aptamer, 31 previously developed to specifi cally bind to CEM cells (T cell line, human acute control, was chosen for the study ( Figure 5 12 A ). According to flow cytometry histograms, an obvious shift (binding) wa s observed for CEM cells (target cells ), but only a negligible shift was noticed for Ramos cells (control cells ) ( Figure 5 12 B ). More importantly, even though much fewer engineered Fe Fe 3 O 4 CSNPs (5 nM, 28 aptamers per functionalized nanoparticle) were use d compared to free aptamers (200 nM), a
131 much larger shift was achieved using modified Fe Fe 3 O 4 CSNPs due to the multivalent effect. The affinity of a ligand to its receptor is highly dependent on its valency (the number of sites available for receptor atta chment). Therefore, the presence of multiple aptamers on the surface of functionalized Fe Fe 3 O 4 CSNPs resulted in greater cooperation, thereby enhancing the binding affinity to target cancer cells. With the FITC labeled chimeric aptamer, the modified Fe Fe 3 O 4 CSNPs can be used as specific fluorescence imaging agents with excellent sensitivity. Explore the Potential of Modified Nanocrystals for Magnetic Resonance Imaging MRI is one of the best noninvasive imaging modalities because of its ability to provide a large amount of spatial and temporal information using various contrast agents, especially iron oxide nanocrystals. 81 253 255 Therefore, we also tested the potential of using engineered Fe Fe 3 O 4 CSNPs as T 2 (transverse relaxation time) contrast agents. Based on a self amplifying proximity assay, 91 when multiple modified Fe Fe 3 O 4 CSN Ps bind to the receptors on their target cancer cells, they act cooperatively to form micrometer scale clusters, thereby enhancing the net transverse relaxation of neighboring protons and leading to a darker image. T 2 weighted MRI was taken for both CEM ce lls and Ramos cells treated with functionalized Fe Fe 3 O 4 CSNPs ( Figure 5 12 C ). Similar darkness was observed for engineered Fe Fe 3 O 4 CSNPs incubated with buffer only and Ramos cells. However, a significantly darker spot was obtained for modified Fe Fe 3 O 4 C SNPs mixed with CEM cells, as a result of their highly specific interaction, demonstrating that functionalized Fe Fe 3 O 4 CSNPs can also serve as selective contrast agents with high performance.
132 Comparison B etween Current Method and Traditional Polymer Syste ms Compared to traditional polymer systems used to produce biocompatible nanocrystals from hydrophobic ones, like polyethylene glycol (PEG), the new method demonstrated here provides a more time and labor efficient single step surface engineering approach : the DNA corona not only renders the functionalized nanoparticles with excellent water solubility but also furnishes them with tunable specific targeting. However, multiple steps are needed to accomplish the phase transfer of hydrophobic nanocrystals usin g traditional polymer systems, and additional procedures are further required for conjugating targeting ligands. In addition, without the necessity of a complicated organic synthesis, all these chimeric DNA molecules can be synthesized on a fully automated DNA/RNA synthesizer. If desired, different modifiers, including commercially available organic dyes, functional groups, therapeutic agents, or even short polymers (e.g., PEG), can be introduced at any location of the sequence during synthesis. Conclusion s To summarize, we have developed a one step facile surface engineering approach for hydrophobic nanocrystals using chimeric DNA molecules. This method is simple as well as efficient and can be readily adapted to a broad range of nanocrystals with variable size, composition, and morphology. Engineered nanocrystals possess excellent dispersity in biological fluids with minimal aggregation and long term stability. In particular, this novel surface engineering approach equips modified nanocrystals with designe r molecular recognition to various molecular addresses, varying from nucleic acids to cancer cells. Based on all these superior features, we believe that this newly
133 developed surface engineering approach will greatly facilitate the use of nanocrystals in m any biomedical applications.
134 Table 5 1. Detailed sequence information and CPG selection for amphiphilic DNA. (CPG = controlled pore glass) Name Sequence CPG Lipid T5 Lipid TTT TT T Lipid T10 Lipid TTT TTT TTT T T Lipid T20 Lipid TTT TTT TTT TTT TTT TTT TT T Lipid T40 Lipid TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT T T Lipid T60 Lipid TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT T Lipid 20 Lipid TTA CTC GAG GGA TCC TAG TC FITC FITC cDNA Biotin GAC TAG GAT CCC TCG AGT AA A Lipid PEG s gc8 Lipid (PEG) 4 ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GA FITC FITC s gc8 ATC TAA CTG CTG CGC CGC CGG GAA AAT ACT GTA CGG TTA GA FITC 3 FITC Table 5 2. DLS and zeta potential measurements of Fe Fe 3 O 4 CSNPs engineered with chimeric DNA molecules of different lengths and sequence information. ( Fe Fe 3 O 4 CSNP = Fe Fe 3 O 4 core shell nanoparticle) Name Hydrodynamic diameter (nm) Zeta poten tial (mV) Lipid T5 19.3 1.8 9.76 1.99 Lipid T10 23.6 1.4 15.07 0.71 Lipid T20 27.2 7.5 30.17 0.57 Lipid 20 28.1 3.4 29.55 0.93 Lipid T40 42.8 0.3 34.05 1.32 Lipid T60 55.9 3.6 40.30 0.57
135 Figure 5 1. Strateg y for o ne s tep s urface e ngineering of h ydrophobic n anocrystals with d esigner molecular r ecognition Hydrophobic interactions between the surfactant ligands (purple) on the nanocrystal surface and the diacyllipids (blue) in the chimeric DNA molecules lead t o spontaneous assembly, encapsulating individual nanocrystals inside the diacyllipid core and leaving the oligonucleotide (multi color) corona outside to confer excellent water solubility and tunable molecular recognition. The molecular structure of the ch imeric DNA molecule is shown at the bottom.
136 Figure 5 2. Characterization of chimeric DNA molecule engineered Fe Fe 3 O 4 CSNPs. TEM images of Fe Fe 3 O 4 CSNPs A) before and B ) after chimeric DNA molecule modification in he xane and water, respectively. C ) Sol vent dispersity of Fe Fe 3 O 4 CSNPs before and after chimeric DNA molecule functionalization. The as prepared Fe Fe 3 O 4 CSNPs are hydrophobic and disperse in hexane, while the functionalized Fe Fe 3 O 4 CSNPs are hydro philic and disperse in water. D ) Magnetic se paration of engineered Fe Fe 3 O 4 CSNPs. Lipid T20 was used as the chimeric DNA molecule her e Scale bars: 50 nm. (Fe Fe 3 O 4 CSNP = Fe Fe 3 O 4 core shell nanoparticle ) Figure 5 3. DLS measurements of modified Fe Fe 3 O 4 CSNPs in water (red) and unmodified Fe F e 3 O 4 CSNPs in hexane (black). Both unmodified and modified nanoparticles had a relatively narrow size distribution. In addition, the average diameter increase from 16.2 nm of unmodified nanoparticles to 27.2 nm of modified nanoparticles indicated a success ful surface engineering. The chimeric DNA molecule used here was lipid T 20 (Fe Fe 3 O 4 CSNP = Fe Fe 3 O 4 core shell nanoparticle)
137 Figure 5 4. UV Vis spectra of modified Fe Fe 3 O 4 CSNPs in water (red) and unmodified Fe Fe 3 O 4 CSNPs in hexane (black). The peak in the red curve is the characteristic absorption peak of DNA around 260 nm. The chimeric DNA molecule used here was lipid T 20 (Fe Fe 3 O 4 CSNP = Fe Fe 3 O 4 core shell nanoparticle) Figure 5 5. FT IR spectra of modified (red) and unmodified (black) Fe Fe 3 O 4 CSNPs. The chimeric DNA molecule used here was lipid T 20 (Fe Fe 3 O 4 CSNP = Fe Fe 3 O 4 core shell nanoparticle)
138 Figure 5 6. Fluorescence intensity and the number of ligand per engineered nanoparticle as a function of chimeric DNA molecule concentration for Fe Fe 3 O 4 CSNPs modified with lipid T 20 (Fe Fe 3 O 4 CSNP = Fe Fe 3 O 4 core shell nanoparticle) Figure 5 7. Fe Fe 3 O 4 CSNPs treated with different concentrations of lipid T 20 Fe Fe 3 O 4 CSNPs modified with adequate chimeric DNA molecules ( 10 M) are soluble A) upon adding water whereas these ones engineered with insufficient ligands need B) vigorous votex (5 M) or C) even sonication (2.5 M) (Fe Fe 3 O 4 CSNP = Fe Fe 3 O 4 core shell nanoparticle)
139 Figure 5 8. Fe Fe 3 O 4 CSNPs treated with c himeric DNA molecule s of different lengths and sequence information. (Fe Fe 3 O 4 CSNP = Fe Fe 3 O 4 core shell nanoparticle) Figure 5 9. Surface engineering of various hydro phobic nanocrystals with different size, composition, and morphology with chimeric DN A molecule TEM images of A ) Fe 3 O 4 NPs (7 nm ); B ) Fe 3 O 4 NPs (15 n m ); C) CdS/ZnS QDs ; D ) Au NPs; E ) Fe Pt NRs; and F ) Au Fe 3 O 4 DNPs after chimeric DNA molecule engineering in water. Lipid T 20 was used as the chimeric DNA molecule here. Scale bars: 50 nm. (Fe 3 O 4 NP = Fe 3 O 4 nanoparticle, CdS/ZnS QD = CdS/ZnS quantum dot, Au NP = Au nanoparticle Fe P t NR = Fe Pt nanorod Au Fe 3 O 4 DNP = Au Fe 3 O 4 dimer nanoparticle ) L T5 L T10 L T20 L 20 L T40 L T60
140 Figure 5 10. Check the presence of empty micelles. TEM images of A ) as synthesized 7 nm Fe 3 O 4 NPs in hexane, B ) lipid T20 functionalized 7 nm Fe 3 O 4 NPs in water, and C ) lipid T20 only in water after negative staining by 2% uranyl acetate. With the negative staining technique, chimeric DNA molecules can be visualized as white circles in B ) or whi te dots in C ). Scale bar s : 100 nm. ( Fe 3 O 4 NP = Fe 3 O 4 nanoparticle) Figure 5 11. Hybridization between chimeric DNA molecul e engineered Fe Fe 3 O 4 CSNPs with their cDNA. A ) Schematic illustration of the hybridization reaction. B ) Confocal laser scanning m icroscopy and C ) SEM images of cDNA Si MSs treated with lipid 20 and lipid T 20 functionalized Fe Fe 3 O 4 CSNPs. Both lipid 20 and lipid T 20 were labeled with FITC. cDNA is perfectly complementary to lipid 20 Scale bars in B C ): 600 nm. ( Fe Fe 3 O 4 CSNP = Fe Fe 3 O 4 core shell nanoparticles cDNA = complementary DNA, SiMS = silica microsphere)
141 Figure 5 12. Specific b inding of chimeric DNA molecule engineered Fe Fe 3 O 4 CSNPs to target cancer cells. A ) Schema tic illustration of the binding event. B ) Flow cytometry histograms of CEM cells (target cells ) and Ramos cells (control cells ) incubated with buffer only, apt amer (200 nM) and engineered Fe Fe 3 O 4 CSNPs (5 nM). PI CEM and PI Ramos are CEM cells and Ramos c ells C ) T 2 weig hted MRI image of engineered Fe Fe 3 O 4 CSNPs only (top), CEM cells (middle) and Ramos cells (bottom) treated with engineered Fe Fe 3 O 4 CSNPs. Lipid PEG s gc8 was used as the chimeric DNA molecule here ( Fe Fe 3 O 4 CSNP = Fe Fe 3 O 4 core shell nanoparticle)
142 CHAPTER 6 SUMMARY AND FUTURE DIRECTIONS Funct ional Nucleic Acid Incorporated Nanomaterials for Bioanalytical and Biomedical Applications Nucleic acids, no matter found in nature or developed in vitro play significant roles in different science and engineering branches. Numerous functional nucleic acid probes and structures have been designed and constructed for a broad spectrum of applications. One of the most important properties of functional nucleic acids is their super b molecular recognition capability through Watson Crick base paring or binding pocket structure form ation By introducing functional nucleic acids to nanomaterials with unique physical and chemical properties, the corresponding conjuga tes functional nucleic acid incorporated nanomaterials provide even wider applications than any of the component alone. Functional nucleic acid incorporated nanomaterials offer several improvements over their component parts : (1) Functional nucleic aci ds can provide nanomaterials with tunable molecular recognition ability ; (2) Nanomaterials can enhance the molecular recongtion capability of functional nucleic acids through cooperative interaction ; (3) Functional nucleic acid incorporated nanomaterials n ot only preserve the character istic s of th eir component s but also produce new features which don t exist otherwise For example, with the help of nanomaterials, functional nucleic acids can escape from nuclease digestion. 53 The overall direction of this doctoral research has been dedicated to develop functional nucleic acid incorporated nanomaterials for bioanalytical and biomedical applications, including early disease diagnosis, targeted drug delivery, and effective canc er therapy. Four major projects have been presented: (1) Smart multifunctional nanostructure for targeted cancer chemotherapy and magnetic resonance imaging; (2)
143 Semiquantification of ATP in live cells using nonspecific desorption of DNA from graphene oxid e as the internal reference ; (3) DNA micelle flares for intracellular mRNA imaging and gene therapy; (4) One step facile surface engineering of hydrophobic nanocrystals with designer molecular recognition. Smart M ultifunction al N anostructure for T argeted C ancer C hemotherapy and M agnetic R esonance I maging In cancer chemotherapy, a high level of anticancer drugs at the tumor site mean that sufficient drug molecules will automatically be uptaken by the target cancer cells to effectively ki ll them. Therefore, other than tumor targeting, the ability to induce efficient uptake of anticancer drugs is an equally important factor to consider when choosing the proper targeting ligand. In this work, we constructed a smart multifunctional nanostruct ure (SMN) from a porous hollow magnetite nanoparticle (PHMNP) loaded with anticancer drug doxorubicin (DOX) a heterobifunctional PEG ligand, and an aptamer. Instead of using an aptamer with targeting ability only we chose s gc8 aptamer with specific bindi ng followed by efficient internalizat ion to CEM cells (target cells) Using this strategy, enhanced binding and uptake of SMNs to CEM cells rather t han Ramos cells (control cells) was observed, resulting in increased therapeutic efficiency and decrease d IC 50 for DOX In addition, SMN showed great potential for magnetic resonance imaging (MRI) Semiquantification of ATP in L ive C ells U sing N onspecific D esor ption of DNA from G raphene O xide as the I nternal R e ference While there has been research to understand intracellular events, very few studies have dealt with quantitation or semiquantitation in livin g cells. In this work, we made effective use of a nanomaterial specifically graphene oxide (GO) both as a quencher and a carrier for intracellular delivery. I n addition, this GO also served as the
144 carrier for reference probes for fluorescent imaging. An ATP aptamer molecular beacon (AAMB) was adsorbed on GO to form a double quenching platform. The AAMB/GO spontaneously enter ed cells, and then AAMB was released and opened by intracellular ATP. The resulting fluorescence recovery was used to perform ATP live cell imaging with greatly improved background and signaling. Moreover, a control ssDNA, which was released nonspecifically from GO by nontarget cellular prote ins, could serve as an internal reference for ATP semiquantification inside living cells using the AAMB to control intensity ratio This approach can serve as a method for intracellular delivery and quantitative analysis. DNA M icelle F lares for I ntracellul ar mRNA I maging and G ene T herapy Nucleic acid probes, which possess simple synthesis, facile modification, and selective binding, hold great promise for disease diagnosis and gene therapy. However, their potentials have not been fully realized due to : (1) A s negatively charged hydrophilic biomacromolecules, nucleic acid probes cannot freely traverse the cell membrane, thus requiring additional instrument or materials for efficient internalization; (2) N ucleic acid probes can be unstable even after successfu l cellular delivery because of endogenous nuclease digestion, leading to high false positive signals or decreased therapeutic efficiency. To address these problems, we developed a novel nucleic acid probe, named molec ular beacon micelle flare (MBMF ), with efficient self delivery and enhanced enzymatic stability, and successfully utilized it for combined messenger RNA (mRNA) detection and gene therapy. MBMFs were prepared by diacyllipid molecular beacon self assembly, not requiring any materials of potential biohazard. Just like pyrotechnic flares that produce brilliant lig ht when activated, MBMFs underwent a
145 significant burst of fluorescence enhancement upon target binding. This hybridization event subsequently induce d gene silencing, leading to cancer cell apoptosis. One S tep F acile S urface E ngineering of H ydrophobic N anocrystals with D esigner M olecular R ecognition High quality nanocrystals have demonstrated substantial potential for biomedical applications. However, being generally hydrophobic, their use ha s been greatly limited by complicated and inefficient surface engineering that often fails to yield biocompatible nanocrystals with minimal aggregation in biological fluids and active targeting toward specific biomolecules. Using chimeric DNA molecules, we developed a one step facile surface engineering method for hydrophobic nanocrystals. The procedure is simple and versatile, generating individual nanocrystals with multiple ligands. In addition, the resulting nanocrystals can actively and specifically tar get various molecular addresses, varying from nucleic acids to cancer cells, by changing the DNA molecules from random sequences to aptamers. Together, the strategy developed here holds great promise in generating the critical technologies needed for biome dical applications of nanocrystals. In summary, this research has mainly focused on developing functional nucleic acid incorporated nanomaterials for early disease diagnosis, targeted drug delivery, and effective cancer therapy. Successful outcomes from th ese studies will greatly expand the applications of functional nucleic acid incorporated nanomaterials in bioanalytical and biomedical fields In addition, this line of research will also provide useful input for future design and construction of functiona l nucleic acid incorporated nanomaterials with superior properties.
146 Future Directions In vivo Applications of Smart Multifunctional Nanostructure and DNA/Graphene Oxide System We have successfully demonstrated the applica tions of SMN for targeted chemother apy and magnetic resonance imaging as well as the a pplication of a DNA/ GO system for ATP semiquantification at the cellular level in vitro I n the next step, we will explore the performance of both systems in vivo using mouse tumor xenograft model For SM N prevention of DOX release under physiological pH (leaking) is desired before any in vivo experiment. This can be realized by completely block ing the pores of PHMNP s using a layer of an acid sensitive and bi ocompatible material such as CaCO 3 or hydroge l s. Regarding the DNA/ GO system, after prov ing its application for semiquantification of ATP in vivo we will expand the application of this system to detect disease related proteins using their corresponding aptamers generated from Syste matic Evolution of Ligands by EXponential e nrichment ( SELEX ) All in One DNA Micelles for Cancer Targeting, Imaging, and Therapy DNA micelles are self assembled from amphiphilic oligonucleotides. T he driving force for this bottom up self assembly process is nonspecific hydr ophobic interaction, providing the foundation for preparing mixed DNA micelles from amphiphilic oligonucleotides with different functionalities. In addition, there also exists the ability to introduce hydrophobic nanoparticles and anticancer drugs to the h ydrophobic core of DNA micelles to provide them with unique magnetic, optical, thermal and therapeutic properties. Therefore, the future direction of DNA micelle related research will be the construction of all in one DNA micelles capable of cancer target ing, imaging, and therapy to provide a useful multi modal tool in the battle against cancer.
147 Cancer targeting will be achieved through the use of diacyllipid aptamer conjugates (L aptamers) Cancer imaging will be realized by diacyllipid molecular beacon c onjugates (L MBs) or the encapsulation of optical nanomaterials, for instance, semiconductor quantum dots (QDs) into the core of DNA micelles. Cancer therapy will be enforced by antisense therapy using L MBs chemotherapy using loaded anticancer drugs, hy perthermia therapy using embedded magnetic nanoparticles, or a combination of the se methods The first trial of all in one DNA micelles will be th o se assembled from L aptamers and L MBs According to our previous results, the cancer targeting, imaging, an d therapy capability of these DNA micelles should be relatively easy to demonstrate at cellular level in vitro For in vivo experiments, the stability of these DNA micelles in blood under extremely diluted condition s thereby mimicking the real situation i n an animal model, will be evaluated. If the distortion or destruc tion of micellar structures is observed, core cross linked DNA micelles will be designed and synthesized through the following approaches: (1) P olymerization using acrylamide containg diacyl lipid o ligonucleotide conjugates; (2) D isulfide cross linking using diacyllipid oligonucleotide conjugates containing multiple consecutive thiol groups; (3) P hoton initiated core cross linking using diacyllipid oligonucleotide conjugates with multiple cont inuous functional groups capable of polymerization under light irradiation.
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168 BIOGRAPHICAL SKETCH Tao Chen was born in Yichan g, Hubei Province, China She attended Three Gorges Senior High School and won first prize in Hubei Province at Chinese Chemistry Olympiad under the guidance of her favoriate teacher Xin Chen. Then she atte nded Wuhan University to study c hemistry. Supervised by Dr. Jingui Qin and Dr. Zhike He, her undergraduate research was focused on synthesizing two photon absorption molecules for efficient photodynamic therapy and molecular light switches for sensitive nucleic acid det ection. With great interest in c hemistry, Tao came to the United States in the fall of 2008 to work under the mentorship of Dr. Weiho ng Tan. She received her Doctor of Philosophy degree in c hemistry from t he University of Florida in August of 2013.