Chemistry advance in molecular imagingMarch 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 ).

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 3 891 ( 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. 4 457 ( 2004 )

Blasberg R G, Multimodality in vivo molecular-genetic imaging, Bioconjugate Chem 15 1376 ( 2004 )

• 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

Radiology Molecular imagingx-ray findings 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 vivotracking of molecular pathway

?Gene-based personal medicine

• Molecular imaging 之機制1. receptor–ligand model2. antigen–antibody model3. transporter–substrate model4. enzyme–substrate model5. 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

• 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 thymidinekinase ( 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.

• 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 ). Imatinibtargets KIT receptor signaling, and its downstream events involve the regulation of glucose uptake and metabolism.

• Molecular imaging : target identification and chemical / biological probes• Probe 之組成• Linker

Probe A Probe B

• for imaging to couple with target• detector (chemical or biological)；biorecognition性質• Probe A for imaging detector• Optical imaging

1. organic fluorophore (small synthetic fluorescent dyes)2. genetically expressed fluorescent protein (GFP, RFP, etc)3. Luciferase4. luminescent semiconductor nanocrystals (quantum dots, QDs), Cd Se / ZnS

• Magnetic resonance imaging• T1 – agent：Gd–DTPA derivatives• Gd–DOTA derivatives• T2 – agent：superparamagnetic iron oxide（SPIO）

Ａ Ｂ

• 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 DOTA derivatives or peptide derivatives

• 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 SbHg 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

• 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 (TOPO, 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 CdSeQDs 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

3.preparation of water–soluble QDs• ＃silicon / siloxane coating : 用 3–(mecaptopropyl)• trimethoxysilane (MPS) 取代TOPO• ＃bifunctional ligands : 用mercaptoacetie acid,• dimercaptosuccinic acid or dithiothreitol取代TOPO• 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 nm2. high photostability3. tunable size dependent emission peaks

• Recently ,X..Michalet et al., Quantum dots for live cells, in vivo imaging, and diagnostics, Science 307 538 ( 2005 )

• Good review for solubilization and functionalization of quantum dots

• 當四氧化三鐵（Fe3O4）或γ–Fe2O3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（SPIO）。

• 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SPIO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，Fe3O4，150nm），Sinerem（dextran包覆，Fe3O4，30nm），（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

• 氧化鐵一詞所含之化學組成有Fe3O4（magnetite），• γ–Fe2O3（maghemite），β–Fe2O3，α–Fe2O3

（haematite），δ–FeOOH（feroxghite）和γ–FeOOH（lepidocrocite）等，目前用於MRI分子影像造影劑的是γ-Fe2O3/ Fe3O4。

• 奈米γ–Fe2O3之醫學應用受制於其different coating，size uniformity和crystallinity之影響。

• 奈米γ–Fe2O3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H2O2使其充分氧化而得到。常用鹼性化合物有KOH，KOH+1﹪PVA，NaOH，NH3，tetramethyl ammonium hydroxide（TMAOH），TMAOH/NH3等。它的反應包括水解與氧化，反應化學式如下：

• Fe2＋＋2Fe3＋＋8OH－ ─→ Fe3O4（black colloidal particles）＋4H2O

• Fe3O4＋0.25O2＋4.5H2O ─→ 3Fe(OH)3• 2Fe(OH)3 ─→ Fe2O3＋3H2O

• 由於anisotropic dipolar attraction，奈米γ–Fe2O3/ Fe3O4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–Fe2O3/ Fe3O4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

• 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 ( CO )5 complex• ( 5 ) Fe ( acac )3 complex• ( 6 ) Fe ( II )–CM–cellulose complex• 通常使用dextran來包覆奈米γ–Fe2O3/ Fe3O4，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。

• 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：

OH

OHCl C

O

Cl+O C

OCl

OH

ROH

RNH2

Carbamate ester

Carbonate esterO C

OOR

OH

OH

O CO

NHR

(2)用phosgene activation

dextran

O

Oglucose

O

OH

glucoseHO

OHHIO4

-glucose O

O

O

O

O glucose

dextran aldehyde

RNH2

glucose O

O

O

HNR

O glucose

reduced product

NaBH4 glucose O

O

O

NR

O glucose

Schiff base

• 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）。

• 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 10 993 ( 2004 )• Annexin V–CLIO–Cy 5.5• CLIO : 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 15 1062 ( 2004 )

• CdSe/ZnS–γ–Fe2O3–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 4 409 ( 2004 )

• Suggestion• From R. Weissleder’s words: One 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.