Chemistry advance in molecular imaging March 8, 2005 ABSTRACT Zeitsan Tsai

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Chemistry advance in molecular imaging March 8, 2005 ABSTRACT Zeitsan Tsai Molecular imaging is broadly defined as the characterization & measurement of biological processes in vivo at cellular and

1 Chemistry advance in molecular imaging March 8, 2005 ABSTRACT Zeitsan Tsai Molecular imaging is broadly defined as the characterization & measurement of biological processes in vivo at cellular and molecular level using imaging techniques. The most widely used imaging modalities are ( 1 ) optical ( fluorescence, bioluminescence, luminescent semiconductor nanocrystals ), ( 2 ) magnetic resonance ( contrast agents: T1 and T2 agents ), and ( 3 ) radionuclides ( PET and SPECT ). The basic approach to molecular imaging includes target identification & development of chemical / biological probes. Those probes should be adapted to the imaging modalities mentioned above. Recently chemical probes of both superparamagnetic iron oxide ( or so called monocrystalline iron oxide nanoparticles and so on ) and luminescent semiconductor nanocrystals ( or so called quantum dots ) are emerging out and prevailing for both MR and optical imaging, respectively, after coupled with the biological specific to the desired target via appropriate linker ( such as 1 ethyl 3 (3 dimethylaminopropyl)carbodiimide,edac ).

2 The syntheses of both chemical probes will be reviewed and presented. Also some examples of both MR and optical molecular imaging will be presented. Reference: Hyeon T et al, Ultra- large- scale syntheses of monodisperse nanocrystals, Nature Mat ( 2004 ) Hyeon T, Chemical synthesis of magnetic nanoparticls, Chem Commun 927 ( 2003 ) West C M L et al, The potential of positron-emission tomography to study anticancer-drug resistance, Nature Rev ( 2004 ) Blasberg R G, Multimodality in vivo molecular-genetic imaging, Bioconjugate Chem ( 2004 )

3 Molecular imaging is broadly defined as the characterization & measurement of biological processes in vivo at cellular and molecular level using imaging techniques (Dr. Peter Shin-Hwa Yeh) The basic approach to molecular imaging includes target identification & development of chemical / biological probes

4 Radiology x-ray findings Molecular imaging chemical or biological probe specific to a molecular target diagnosis with pathological confirmation validated by image-guided in vitro tissue analysis clinical useful information (pattern recognition) useful information for in vivo tracking of molecular pathway? Gene-based personal medicine

5 Molecular imaging 之機制 1. receptor ligand model 2. antigen antibody model 3. transporter substrate model 4. enzyme substrate model 5. complex or hybrid model Cellular physiologic activities : metabolism, hypoxia, proliferation, apoptosis, angiogenesis, response to infection, multiple drug resistance 分子影像的可能運用 Image guided biopsy Imaging for therapy selection & monitoring Image guided radio therapy Imaging tumor proliferation

6 Molecular imaging strategies Direct imaging strategies: imaging the target directly with a target specific probe. The resultant image of the probe localization and concentration ( signal intensity ) is directly related to its interaction with the target molecule ( which can be a protein or RNA or DNA ) For example: imaging of the vascular endothelial growth factor ( VEGF ) using I 124 labeled anti VEGF monoclonal antibody i.e. I 124 VG76e. Indirect imaging strategies: widely used is reporter gene imaging. For example: The herpes simplex virus type 1 thymidine kinase ( HSV1 tk ) reporter gene linked to the promoter of an endogenous gene of interest. The PET tracer I 124 FIAU is used as a reporter probe. FIAU is an analogue of ganciclovir, which is used in the treatment of herpes simplex virus infection. FIAU traps the reporter-gene product, which is produced when the endogenous gene of interest is transcribed.

7 Surrogate imaging or biomarker: assessing downstream effects of one or more endogenous molecular-genetic processes. For example: The F 18 FDG PET imaging of early functional changes in glucose metabolism that correlate with the tumour response to imatinib ( Glivec ). Imatinib targets KIT receptor signaling, and its downstream events involve the regulation of glucose uptake and metabolism.

8 Molecular imaging : target identification and chemical / biological probes Probe 之組成 Linker A B Probe A Probe B for imaging to couple with target detector (chemical or biological);biorecognition 性質 Probe A for imaging detector ptical imaging 1. organic fluorophore (small synthetic fluorescent dyes) 2. genetically expressed fluorescent protein (GFP, RFP, etc) 3. Luciferase 4. luminescent semiconductor nanocrystals (quantum dots, QDs), Cd Se / ZnS Magnetic resonance imaging T1 agent:gd DTPA derivatives Gd DTA derivatives T2 agent:superparamagnetic iron oxide(spi)

9 Nuclear imaging PET radionuclide chemicals:f 18 or I 124 labeled compounds ; SPECT radionuclide chemicals: Tc 99m, In 111, Ga 67 or Cu 64 labeled compounds;dtpa, EDTA, or DTA derivatives or peptide derivatives

10 Semiconductor nanocrystals, knows as quantum dots (QDs) 原子組成 :group Ⅱ & Ⅵ 或 Ⅲ & Ⅴ elements 週期表 Zn S Ga As Cd Se In Sb Hg Te Tl group Ⅱ&Ⅵ (CdSe, CdTe, CdS, ZnSe) group Ⅲ&Ⅴ (InP, InAs) Before 1993, QDs were mainly prepared in aqueous solution with added stability agents (e.g. thioglycerol or polyphosphate); low quality QDs with poor fluorescence efficiencies and large size variations (relative standard deviation, RSD, > 15%) In 1993, Bawendi and coworker synthesized highly luminescent CdSe QDs with a high temperature organometallic procedure ; RSD<5% but fluorescence quantum yields were still relatively low (~10%)

11 1996~1997, 發展 ZnS capped CdSe nanocrystals, 以增加 CdSe 之 quantum yields 至 40~50% 合成方法 : 在 CdSe / tri n octylphosphine oxide (TP, a high boiling point and coordinating solvent) 溶液中, 加入 Zn(CH3)2/S 溶液, 得到 CdSe core 上被覆一層 ZnS 2001, ten distinguishable emission colors of ZnS capped CdSe QDs excited with a near UV lamp : 443, 473, 481, 500, 518, 543, 565, 587, 610 and 655 nm QDs optical properties : ZnS capped CdSe Full width at half maximum (FWHM) : 13 nm Lifetimes : 5 ns, 20~30 ns (dominant), 80~200 ns Molar extinction coefficient : 105~106 M-1 cm-1 ZnS capped CdSe QDs surface chemistry morphologically, QDs are not smooth spherical particles, but are faceted with many plane and edges possess negative charge or positive charge

12 3.preparation of water soluble QDs #silicon / siloxane coating : 用 3 (mecaptopropyl) trimethoxysilane (MPS) 取代 TP #bifunctional ligands : 用 mercaptoacetie acid, dimercaptosuccinic acid or dithiothreitol 取代 TP Bioconjugation The surface area of a single QD is large enough for linking to multiple biomolecules: Two to 5 protein molecules and 50 or more small molecules (such as oligonucleotides or peptides) may be conjugated to a single 4 nm QD Luminescent semiconductor quantum dots (QDs) 之优勢 1. sharp emission spectra with full width at half-maximum (FWHM)~ 25 nm 2. high photostability 3. tunable size dependent emission peaks Recently,X..Michalet et al., Quantum dots for live cells, in vivo imaging, and diagnostics, Science ( 2005 ) Good review for solubilization and functionalization of quantum dots

13 當四氧化三鐵 (Fe34) 或 γ Fe23 的氧化鐵的粒徑小於 35nm 後, 即達到奈米級時, 其磁性之表現即不同, 即每個氧化鐵顆粒之磁性即成為一個 Single domain, 在有外加磁場時才是磁鐵 (ferromagnets), 而無外加磁場時卻是順磁性 (paramagnetism), 不再保有磁性, 此種小於 35nm 的奈米氧化鐵具有超磁鐵 (superparamagnets) 特性一般稱為 superparamagnetic iron oxide(spi) 奈米氧化鐵之醫學應用, 包括 : (1) 當作 MRI 對比劑 ; 利用原理是這種 SPI 經由循環不會立即被網狀內皮系 (reticuloendothetial system) 移除, 因此有較長之血液半生期和較寬廣之身體分佈, 商品有 Lumirem ( 氧化矽包覆,300nm),Endorem(dextran 包覆, Fe34,150nm),Sinerem(dextran 包覆,Fe34, 30nm),(Brigger,2002) (2) controlled drug delivery (3) bioprocesssing;in vitro cell separation (4) magnetic intracellular hyperthermia (5) 作 MRI 分子影像造影劑

14 氧化鐵一詞所含之化學組成有 Fe34(magnetite), γ Fe23(maghemite),β Fe23,α Fe23 (haematite),δ FeH(feroxghite) 和 γ FeH (lepidocrocite) 等, 目前用於 MRI 分子影像造影劑的是 γ- Fe23/ Fe34 奈米 γ Fe23 之醫學應用受制於其 different coating,size uniformity 和 crystallinity 之影響奈米 γ Fe23 之製備方法, 是用 FeCl2 和 FeCl3, 在鹼性溶液裡, 使其產生化學反應, 並且再通氧氣或再加入 H22 使其充分氧化而得到常用鹼性化合物有 KH, KH+1 % PVA,NaH,NH3,tetramethyl ammonium hydroxide(tmah),tmah/nh3 等它的反應包括水解與氧化, 反應化學式如下 : Fe2++2Fe3++8H- Fe34(black colloidal particles)+4h2 Fe H2 3Fe(H)3 2Fe(H)3 Fe23+3H2

15 由於 anisotropic dipolar attraction, 奈米 γ Fe23/ Fe34 很容易聚積成塊 (clustters), 而失去原有 single domain 之磁性因此要加入穩定劑或將其包覆 (coating) 常用之穩定劑有 :sodium citrate,oleic acid,lauric acid,cationic surfactants( 如 tetraoctyl ammonium bromide )sodium chloride 等常用奈米 γ Fe23/ Fe34 之規格為 0.9 % NaCl solution containing 11.6mg Fe/ml and 75mM sodium citrate(center for molecular imaging reserrch,mgh,boston), 要放在 4 冰櫃保存 Recent advance in preparation of iron oxide nanocrystals Goal: ( 1 ) good size control ( 2 ) narrow size distribution ( 3 ) good crystallinity ( 4 ) dispersable nanocrystal

16 Using non hydrolytic precursors; thermal decomposition in high boiling point organic solvents ( 1 ) Fe ( oleate )3 : 1 hexadecane ( b.p.274 C ) ; 5 nm crystal octyl ether ( b.p 287 C ) 9 nm crystal 1 octadecene ( b.p. 317 C ) 12 nm crystal 1 eicosene ( b.p. 330 C ) 16 nm crystal trioctylamine ( b.p. 365 C ) 22 nm crystal ( 2 ) Fe ( II ) alginate complex ( 3 ) Fe ( cuferron ) 3 complex ( 4 ) Fe ( C )5 complex ( 5 ) Fe ( acac )3 complex ( 6 ) Fe ( II ) CM cellulose complex 通常使用 dextran 來包覆奈米 γ Fe23/ Fe34, 其他之包覆化合物包括 reduced dextran(paul,2004),amylose starch(veigta,2000), liposome(bulte,1999),meso 2,3 dimercaptosuccinic acid (Wilhelm,2003),poly(ethylene glycol),polystyrene(wang, 2003),silica amphorous polymers 等

17 為了要達成生物接枝, 在包覆分子與特定生物分子之間, 要有連接劑 (linker 或 spacer 或 bridger), 包括 diols,diamines,diminde,dicarboxylic acid 等, 如 1 ethyl 3 (3 dimethylaminopropyl) carbodiimide (EDAC) Mehvar(2000) 曾討論 dextran 之處理, 包括 : (1) 用 periodate 方法 : glucose H H H dextran glucose glucose HI4- dextran aldehyde glucose RNH 2 glucose NaBH4 glucose glucose glucose HNR reduced product NR Schiff base (2) 用 phosgene activation H + H Cl C Cl H C Cl RH RNH 2 H C R Carbonate ester H C NHR Carbamate ester

18 化學處理過之 dextran, 再與 drug, proteins/enzyme 或 imaging agents 連接, 而達到預定之目的, 如治療造影診斷等已知之奈米氧化鐵生物接枝劑如下 : 1.annexinV(Schellenberger,2002) 2.lactoferrin 和 ceruloplasmin(gupta,2004) 3.transferrin(Hogemann,2000)

19 Some examples of CdSe/ZnS quantum dot and superparamagnetic iron oxide CdSe/ZnS DHLA B16F10 melanoma cells Linker: dihydroxylipoic acid ( DHLA ) Detection: fluorescence emission scanning microscopy Small animal: syngenetic C57BL/6 mice Purpose: tracking metastatic tumor cell extravasation Reference: Nature Medicine ( 2004 ) Annexin V CLI Cy 5.5 CLI : cross-linked iron oxide Cy 5.5 : near infrared ray fluorescent dye Annexin V : to bind to phosphatidylserine ( PS ) Linkers : SPDP: N succinimidyl 3 (2 pyridyldithio) propionate; SATA: N succinimidyl 3 (2 pyridylthio) propionate (S acetylthioacetate) Detection: fluorescence microscopy, MRI Purpose: specific for apoptotic Jurkat T cells Reference: Bioconjugate Chem ( 2004 )

20 CdSe/ZnS γ Fe23 mouse anticycline E antibody Linkers : DMSA : dimercaptosuccinic acid; EDAC : 1 ethyl 3 (3 dimethylaminopropyl) carbodiimide Detection: fluorescence microscopy Purpose: to separate MCF 7 breast cancer cells from serum solution using magnetic separation Reference: Nano Letters ( 2004 )

21 Suggestion From R. Weissleder s words: ne of the key aspects of radiology s engagement in molecular imaging will be the recruitment of molecular biologists, cell biologists, and synthetic chemists as well as radiochemists to enhance specialties. This is to reflect both target identification and chemical/biological probe preparation.

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