Name: investigations on the ga(iii) complex of eob-dtpa and its 68ga radiolabeled analogue
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Description: Watch this Scientific Journal Video about Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue at JoVE.com

The authors have no acknowledgements.

1. Preparation of EOB-DTPA and Ga[EOB-DTPA]
Caution: Please consult all relevant material safety data sheets (MSDS) of the used organic solvents, acids and alkalines before use. Perform all steps in a fume hood and use personal protective equipment (safety glasses, gloves, lab coat).
<ol>
	<li>Isolation of EOB-DTPA from gadoxetic acid
	<ol>
		<li>Put 3 ml of 0.25 M gadoxetic acid injectable solution into a flask. Add 500 mg (5.6 mmol) of oxalic acid to the stirred solution.</li>
		<li>After stir...

<ol>
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EOB-DTPA is accessible through a multi-step synthesis33 but may just as well be isolated from available contrast agents containing gadoxetic acid. For this purpose, the central Gd(III) ion can be precipitated with an excess of oxalic acid. After removing Gd(III) oxalate and oxalic acid the ligand can be isolated by precipitation in cold water at pH 1.5. However, in order to enhance yields column chromatography of the filtrate can be performed instead or as a follow-up procedure. Either method yields the analyt...

Gadoxetic acid, a common name for the Gd(III) complex of the ligand EOB-DTPA1, is a frequently used contrast agent in hepatobiliary magnetic resonance imaging (MRI).2,3 Due to its specific uptake by liver hepatocytes and high percentage of hepatobiliary excretion it enables the localization of focal lesions and hepatic tumors.2-5 However, certain limitations of the MRI technique (e.g., toxicity of the contrast agents, limited applicability in patients with claustrophobia or metal implants) call for an alternative diagnostic tool.
Positron emission tomography (PET) is a molecular imaging method, wherein a small amount of a radioactive substance (tracer) is administered, upon which its distribution in the body is recorded by a PET scanner.6 PET is a dynamic method that allows for high spatial and temporal resolution of images as well as quantification of the results, without having to deal with the side-effects of MRI contrast agents. The informative value of the obtained metabolic information can be further increased by combination with anatomical data received from additional imaging methods, as most commonly achieved by hybrid imaging with computed tomography (CT) in PET/CT scanners.
The chemical structure of a tracer suitable for PET must include a radioactive isotope serving as positron emitter. Positrons have a short life-span since they almost immediately annihilate with electrons of the atom shells of surrounding tissue. By annihilation two 511 keV gamma photons with opposite direction of movement are emitted, which are recorded by the PET scanner.7,8 To form a tracer, PET nuclides may be bound covalently to a molecule, as is the case in 2-deoxy-2-[18F]fluoroglucose (FDG), the most extensively used PET tracer.7 However, a nuclide may also form coordinative bonds to one or several ligands (e.g., [68Ga]-DOTATOC9,10) or be applied as dissolved inorganic salts (e.g., [18F] sodium fluoride11). Altogether, the structure of the tracer is crucial as it determines its biodistribution, metabolism and excretion behavior.
A suitable PET nuclide should combine favorable characteristics like convenient positron energy and availability as well as a half-life adequate for the intended investigation. The 68Ga nuclide has become an essential force in the field of PET over the last two decades.12,13 This is mainly due to its availability through a generator system, which allows on-site labeling independently from the vicinity of a cyclotron. In a generator, the mother nuclide 68Ge is absorbed on a column from which the daughter nuclide 68Ga is eluted and subsequently labeled to a suitable chelator.6,14 Since the 68Ga nuclide exists as a trivalent cation just like Gd(III)10,13, chelating EOB-DTPA with 68Ga instead would yield a complex with the same overall negative charge as gadoxetic acid. Accordingly, that 68Ga tracer might combine a similar characteristic liver specificity with the suitability for PET imaging. Although gadoxetic acid is purchased and administered as disodium salt, in the following context we will refer to it as Gd[EOB-DTPA] and to the non-radioactive Ga(III) complex as Ga[EOB-DTPA], or 68Ga[EOB-DTPA] in case of the radiolabeled component for the sake of convenience.
To evaluate their applicability as tracers for PET, radioactive metal complexes need to be examined extensively in in vitro, in vivo or ex vivo experiments first. To determine the suitability for a respective medical problem, various tracer characteristics like biodistribution behavior and clearance profile, stability, organ specificity and cell or tissue uptake need to be investigated. Due to their non-invasive character, in vitro determinations are often performed prior to in vivo experiments. It is generally acknowledged that DTPA and its derivatives are of limited suitability as chelators for 68Ga due to these complexes lacking kinetic inertness, resulting in comparably fast decomposition when administered in vivo.14-20 This is primarily caused by apo-transferrin acting as a competitor for 68Ga in plasma. Nevertheless, we investigated this new tracer concerning its possible application in hepatobiliary imaging, wherein diagnostic information may be provided within minutes post-injection3,4,21-23, thereby not necessarily requiring long-term tracer stability. For this purpose we isolated EOB-DTPA from gadoxetic acid and initially performed the complexation with natural Ga(III), which exists as mixture of two stable isotopes, 69Ga and 71Ga. The complex thus obtained served as non-radioactive standard for the following chelation of 68Ga. We used established methods and simultaneously evaluated their suitability for determining the 68Galabeling efficiency of EOB-DTPA and to investigate the lipophilicity of the new 68Ga tracer and its stability in different media.

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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">primovist</td>
			<td style="width:180px;">Bayer</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">0.25 M</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gallium(III) chloride</td>
			<td style="width:180px;">Sigma-Aldrich Co.</td>
			<td style="width:119px;">450898</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">water (deionized)</td>
			<td style="width:180px;">-</td>
			<td style="width:119px;">-</td>
			<td>&#160;tap water deionizing equipment by Auma-Tec GmbH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">hydrochloric acid 12 M</td>
			<td style="width:180px;">VWR</td>
			<td style="width:119px;">20252.29</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">sodium hydroxide</td>
			<td style="width:180px;">Polskie Odczynniki Chemiczne S.A.</td>
			<td style="width:119px;">810925429</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">oxalic acid</td>
			<td style="width:180px;">Sigma-Aldrich Co.</td>
			<td style="width:119px;">75688</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ethyl acetate</td>
			<td style="width:180px;">Brenntag GmbH</td>
			<td style="width:119px;">10010447</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">silica gel</td>
			<td style="width:180px;">Merck KGaA</td>
			<td style="width:119px;">1.10832.9025</td>
			<td>Geduran Si 60 0,063-0,2 mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TLC silica gel 60 F254</td>
			<td style="width:180px;">Merck KGaA</td>
			<td style="width:119px;">1.16834.0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">methanol</td>
			<td style="width:180px;">VWR</td>
			<td style="width:119px;">20903.55</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ethanol</td>
			<td style="width:180px;">Brenntag GmbH</td>
			<td style="width:119px;">10018366</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">eiethylether</td>
			<td style="width:180px;">VWR</td>
			<td style="width:119px;">23807.468</td>
			<td>stored over KOH plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ammonia solution (25 %)</td>
			<td style="width:180px;">VWR</td>
			<td style="width:119px;">1133.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pH electrode</td>
			<td style="width:180px;">VWR</td>
			<td style="width:119px;">662-1657</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">stirring and heating unit</td>
			<td style="width:180px;">Heidolph</td>
			<td style="width:119px;">505-20000-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pump</td>
			<td style="width:180px;">Ilmvac GmbH</td>
			<td style="width:119px;">322002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">frit</td>
			<td style="width:180px;">-</td>
			<td style="width:119px;">custom design</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NMR spectrometer</td>
			<td style="width:180px;">Bruker Coorporation</td>
			<td style="width:119px;">-</td>
			<td>Ultra Shield 400</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">mass spectrometer</td>
			<td style="width:180px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">elemental analyser</td>
			<td>Hekatech GmbH Analysentechnik</td>
			<td style="width:119px;">-</td>
			<td>EuroVector EA 3000 CHNS</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">deuterated water D2O</td>
			<td style="width:180px;">euriso-top</td>
			<td style="width:119px;">D214</td>
			<td>99,90 % D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name</td>
			<td style="width:180px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;width:681px;">Material/Equipment required for labeling procedures</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">68Ge/68Ga generator</td>
			<td style="width:180px;">ITG Isotope Technologies Garching GmbH</td>
			<td style="width:119px;">A150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pump and dispenser system</td>
			<td style="width:180px;">Scintomics GmbH</td>
			<td style="width:119px;">-</td>
			<td>Variosystem</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">hydrochloric acid 30 % (suprapur)</td>
			<td style="width:180px;">Merck KGaA</td>
			<td style="width:119px;">1.00318.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">water (ultrapur)</td>
			<td style="width:180px;">Merck KGaA</td>
			<td style="width:119px;">1.01262.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium chloride (suprapur)</td>
			<td style="width:180px;">Merck KGaA</td>
			<td style="width:119px;">1.06406.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium acetate (suprapur)</td>
			<td style="width:180px;">Merck KGaA</td>
			<td style="width:119px;">1.06264.0050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">glacial acetic acid (suprapur)</td>
			<td style="width:180px;">Merck KGaA</td>
			<td style="width:119px;">1.00066.0250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium citrate dihydrate</td>
			<td style="width:180px;">VEB Laborchemie Apolda</td>
			<td style="width:119px;">10782</td>
			<td>&#62;98.5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PS-H+ Cartridge (S)</td>
			<td style="width:180px;">Macherey-Nagel</td>
			<td style="width:119px;">731867</td>
			<td>Chromafix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">apo-Transferrin</td>
			<td style="width:180px;">Sigma-Aldrich Co.</td>
			<td style="width:119px;">T2036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PBS&#160; buffer (tablets)</td>
			<td style="width:180px;">Sigma-Aldrich Co.</td>
			<td style="width:119px;">79382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">human serum</td>
			<td style="width:180px;">Sigma-Aldrich Co.</td>
			<td style="width:119px;">H4522</td>
			<td>from human male AB plasma</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">flasks, columns etc.</td>
			<td style="width:180px;"></td>
			<td style="width:119px;">custom design</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">pH electrode</td>
			<td style="width:180px;">Knick Elektronische Messger&#228;te GmbH &#38; Co. KG</td>
			<td style="width:119px;">765-Set</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">binary pump (HPLC)</td>
			<td style="width:180px;">Hewlett-Packard</td>
			<td style="width:119px;">G1312A (HP 1100)</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">UV Vis detector (HPLC)</td>
			<td style="width:180px;">Hewlett-Packard</td>
			<td style="width:119px;">G1315A (HP 1100)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">radioactive detector (HPLC)</td>
			<td style="width:180px;">EGRC Berthold</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">HPLC C-18-PFP column</td>
			<td style="width:180px;">Advanced Chromatography Technologies Ltd.</td>
			<td style="width:119px;">ACE-1110-1503/A100528</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HPLC glass vials</td>
			<td style="width:180px;">GTG Glastechnik Graefenroda GmbH</td>
			<td style="width:119px;">8004-HP-H/i3&#181;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pipette</td>
			<td style="width:180px;">Eppendorf</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">plastic vials</td>
			<td style="width:180px;">Sarstedt AG &#38; Co.</td>
			<td style="width:119px;">6542.007</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">plastic vials</td>
			<td style="width:180px;">Greiner Bio-One International GmbH</td>
			<td style="width:119px;">717201</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">activimeter</td>
			<td style="width:180px;">MED Nuklear-Medizintechnik Dresden GmbH</td>
			<td style="width:119px;">-</td>
			<td>Isomed 2010</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tweezers</td>
			<td style="width:180px;"></td>
			<td style="width:119px;">custom design</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">incubator</td>
			<td style="width:180px;">Heraeus Instruments GmbH</td>
			<td style="width:119px;">51008815</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">vortex mixer</td>
			<td style="width:180px;">Fisons</td>
			<td style="width:119px;">-</td>
			<td>Whirlimixer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">centrifuge</td>
			<td style="width:180px;">Heraeus Instruments GmbH</td>
			<td style="width:119px;">75003360</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">gamma well counter</td>
			<td style="width:180px;">MED Nuklear-Medizintechnik Dresden GmbH</td>
			<td style="width:119px;">-</td>
			<td>Isomed 2100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">water for chromatography</td>
			<td style="width:180px;">Merck KGaA</td>
			<td style="width:119px;">1.15333.2500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">acetonitrile for chromatography</td>
			<td style="width:180px;">Merck KGaA</td>
			<td style="width:119px;">1.00030.2500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">trifluoroacetic acid</td>
			<td style="width:180px;">Sigma-Aldrich</td>
			<td style="width:119px;">91707</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">TLC radioactivity scanner</td>
			<td style="width:180px;">raytest Isotopenmessger&#228;te GmbH</td>
			<td style="width:119px;">B00003875</td>
			<td>equipped with beta plastic detector</td>
		</tr>
	</tbody>
</table>

investigations on the ga(iii) complex of eob-dtpa and its 68ga radiolabeled analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) complex and protocols for the preparation of its novel non-radioactive, i.e., natural Ga(III) as well as radioactive 68Ga complex. The ligand as well as the Ga(III) complex were characterized by nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and elemental analysis. 68Ga was obtained by a standard elution method from a 68Ge/68Ga generator. Experiments to evaluate the 68Ga-labeling efficiency of EOB-DTPA at pH 3.8&#8211;4.0 were performed. Established analysis techniques radio TLC (thin layer chromatography) and radio HPLC (high performance liquid chromatography) were used to determine the radiochemical purity of the tracer. As a first investigation of the 68Ga tracers&#39; lipophilicity the n-octanol/water distribution coefficient of 68Ga species present in a pH 7.4 solution was determined by an extraction method. In vitro stability measurements of the tracer in various media at physiological pH were performed, revealing different rates of decomposition.

The ligand EOB-DTPA and the non-radioactive Ga(III) complex were analyzed via 1H and 13C{1H} NMR spectroscopy, mass spectrometry and elemental analysis. The results listed in Table 1 and depicted in Figures 1-6 verify the purity of the substances.
Elution of the 68Ge/68Ga generator yielded solutions of 400-600 MBq 68Ga. T...

Watch this Scientific Journal Video about Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue at JoVE.com

Investigations on the GaIII Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

A procedure for the isolation of EOB-DTPA and subsequent complexation with natural Ga(III) and 68Ga is presented herein, as well as a thorough analysis of all compounds and investigations on labeling efficiency, in vitro stability and the n-octanol/water distribution coefficient of the radiolabeled complex.

Investigations on the Ga(III) Complex of EOB-DTPA and Its 68Ga Radiolabeled Analogue

We demonstrate a method for the isolation of EOB-DTPA (3,6,9-triaza-3,6,9-tris(carboxymethyl)-4-(ethoxybenzyl)-undecanedioic acid) from its Gd(III) ...

The overall goal of this procedure is to evaluate a new radioactive gallium-68 complex for molecular imaging. This method can help answer key questions in the field of gallium-68 tracer chemistry such as labeling conditions, tracer stability and lipophilicity. The technique of radioactive labeling is straightforward and allows for fast determination of the results while requiring only minimum amounts of material.To begin this procedure, add three milliliters of 0.25-molar gadoxetic acid injectable solution to a flask. Then add 500 milligrams of oxalic acid to the stirring gadoxetic acid solution. After stirring for one hour, filter the suspension through a frit using reduced pressure.Wash the residue three times with three milliliters of water. Next, combine the aqueous filtrates in a beaker and place a pH electrode in the solution. Add 12 molar hydrochloric acid to the filtrate until the pH is about 0.1.After removing the solvent in vacuo, wash the residue thoroughly with hot ethyl acetate to remove the access oxalic acid. Then dry the residue in vacuo. Once the residue is dry, redissolve it in two milliliters of de-ionized water and then cool the solution in an ice bath.Without removing the ice bath, add 0.5 molar sodium hydroxide drop wise until a colorless, gluey solid is observed. Then remove the water with a pipette. To prepare a 0.11 molar stock solution of gallium trichloride dissolve 1.94 grams of the solid in 100 milliliters of water.Next, dissolve 80 milligrams of EOB-DTPA in 10 milliliters of water. If necessary, heat the solution to achieve complete dissolution. Add 1.4 milliliters of the gallium trichloride stock solution to the EOB-DTPA solution and place a pH electrobe in the beaker.Then add diluted aqueous ammonia solution drop wise until the pH of the solution is approximately 4.1. After removing the solvent in vacuo, place the residue in a flask equipped with a steelhead with a central and parallel side neck. Equip the central neck with a cooling finger and the side neck with a vacuum pump outlet.Heat the residue under reduced pressure. Periodically remove sublimated ammonium chloride from the flask with a slightly wet cloth. Precondition a PSH plus cartridge by flushing it slowly with 1 milliliter of 1 molar hydrochloric acid and subsequently, 5 milliliters of water.Following this, elute the silica column of the generator with four milliliters of 0.05 molar hydrochloric acid and load the gallium-68 eluate into the PSH plus cartridge. Flush the cartridge with five milliliters of water. After drying with air, elute the gallium-68 from the cartridge with one milliliter of five molar acidified sodium chloride.Now add 50 microliters of the gallium-68 eluate and 50 microliters of a previously-prepared 19 micromolar EOB-DTPA stock solution to a vial. Add 300 microliters of buffer to raise the pH to 4.0. Shake briefly and incubate the solution at room temperature for five minutes.Transfer a five to 20 microliter aliquot of the solution to a HPLC vial. Then preform radio HPLC analysis on a reversed phase C18 column. To preform in vitro stability measurements, withdraw samples from the labeling solution containing six to 12 megabecquerel of tracer.Perform radio TLC analysis on 80 millimeter silica gel-coated aluminum plates using 0.1 molar aqueous sodium citrate as eluent. Analyze the plates with a TLC radioactivity scanner. To determine the stability in phosphate buffered saline, add 150 microliters of 10 millimolar PBS stock solution and 60 microliters of 0.1 molar sodium hydroxide to 65 microliters of labeling solution.Mix the solution thoroughly. Following this, transfer a one to five microliter aliquot of the solution to a silica plate for TLC analysis. Immediately store the solution in an incubator at 37 degrees Celsius.Remove aliquots to perform TLC analysis at representative time points over three hours. To determine the stability toward an excess of apotransferrin, add 50 microliters of 10 millimolar PBS stock solution and 430 microliters of 0.1 molar sodium hydroxide to 120 microliters of labeling solution. Add 40 microliters of a 25 milligram per milliliter solution of apotransferrin and thoroughly mix the solution.Next, transfer a one to five microliter aliquot of the solution to a silica plate for TLC analysis. Immediately store the solution in an incubator at 37 degrees Celsius. Remove aliquots to perform TLC analysis at representative time points over three hours.To determine the stability in human serum, add 45 microliters of 0.1 molar sodium hydroxide and 500 microliters of human serum to 25 microliters of labeling solution. After thoroughly mixing the solution, transfer a one to five microliter aliquot to a silica plate for TLC analysis. Immediately store the solution in an incubator at 37 degrees Celsius.Remove aliquots to perform TLC analysis at representative time points over three hours. For determination of the distribution coefficient, add 20 microliters of 10 millimolar PBS stock solution and 170 microliters of 0.1 molar sodium hydroxide to 50 microliters of labeling solution. Transfer 200 microliters of the solution to a plastic V vial and add 200 microliters of n-Octanol.Close the vial and vortex for two minutes, then centrifuge the sample at 1600 times g for five minutes. Following centrifugation, remove triplicates of 40 microliters from the n-Octanol phase and the aqueous phase and place them in separate plastic V vials. Finally, measure the activity of each sample in a gamma well counter for 30 seconds.The ligened EOB-DTPA and the non-radioactive gallium three complex were analyzed via proton and carbon NMR spectroscopy, mass spectrometry and elemental analysis. The labeling procedure results in the formation of the desired tracer gallium-68 EOB-DTPA indicated as a radio HPLC peak exhibiting a retention time of 2.8 minutes. Comparison with the retention time of the cold standard at 220 nanometers confirms successful labeling.The gallium-68 labeling efficiency of EOB-DTPA was investigated by determining the labeling yield as a function of the ligened concentration via HPLC. A representative chromatogram of a TLC plate analyzed with a TLC radioactivity scanner is shown here. To investigate the stability of the tracer, the radiochemical purity over time was determined via TLC.The standardized percentage of intact tracer is depicted as a function of time here. Activity values and subsequent calculation of the distribution coefficient are listed here. Following this procedure, other methods like in vitro experiments can be performed in order to answer additional questions like biodistribution and uptake dynamics.After watching this video, you should have a good understanding of the techniques used to evaluate and analyze gallium-68 tracers for molecular imaging. Don't forget that working with radioactive substances can be harmful. It may only be performed by trained personnel.Safety precautions such as the use of lead shields and tweezers should always be taken while performing this procedure.

investigations-on-gaiii-complex-eob-dtpa-its-68ga-radiolabeled

Research

JoVE Journal

Chemistry

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish1, plants2, 3, arthropods4, 5, fungi6, and bacteria7. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears11, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 &deg;C), and temperature recordings as a function of time were not an available option for these instruments.
We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously9, 10, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.
To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)12. This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)10. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs5, 13. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations14, 15. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber13. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere16.
The Cold Stage System
The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 &mu;m in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).

Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner.

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition ...

Reductive Electropolymerization of a Vinyl-containing Poly-pyridyl Complex on Glassy Carbon and Fluorine-doped Tin Oxide Electrodes

Controllable electrode surface modification is important in a number of fields, especially those with solar fuels applications. Electropolymerization is one surface modification technique that electrodeposits a polymeric film at the surface of an electrode by utilizing an applied potential to initiate the polymerization of substrates in the Helmholtz layer. This useful technique was first established by a Murray-Meyer collaboration at the University of North Carolina at Chapel Hill in the early 1980s and utilized to study numerous physical phenomena of films containing inorganic complexes as the monomeric substrate. Here, we highlight a procedure for coating electrodes with an inorganic complex by performing reductive electropolymerization of the vinyl-containing poly-pyridyl complex onto glassy carbon and fluorine doped tin oxide coated electrodes. Recommendations on electrochemical cell configurations and troubleshooting procedures are included. Although not explicitly described here, oxidative electropolymerization of pyrrole-containing compounds follows similar procedures to vinyl-based reductive electropolymerization but are far less sensitive to oxygen and water.

A procedure for performing reductive electropolymerization of vinyl-containing compounds onto glassy carbon and fluorine doped tin-oxide coated electrodes is presented. Recommendations on electrochemical cell configurations and troubleshooting procedures are included. Although not explicitly described here, oxidative electropolymerization of pyrrole-containing compounds follows similar procedures to vinyl-based reductive electropolymerization.

Controllable electrode surface modification is important in a number of fields, especially those with solar fuels applications. Electropolymerization is ...

Photochemical Oxidative Growth of Iridium Oxide Nanoparticles on CdSe@CdS Nanorods

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide (CdSe@CdS) nanorod photocatalysts. Seeded rods are grown using a colloidal hot-injection method and then moved to an aqueous medium by ligand exchange. CdSe@CdS nanorods, an iridium precursor and other salts are mixed and illuminated. The deposition process is initiated by absorption of photons by the semiconductor particle, which results with formation of charge carriers that are used to promote redox reactions. To insure photochemical oxidative growth we used an electron scavenger. The photogenerated holes oxidize the iridium precursor, apparently in a mediated oxidative pathway. This results in the growth of high quality crystalline iridium oxide particles, ranging from 0.5 nm to about 3 nm, along the surface of the rod. Iridium oxide grown on CdSe@CdS heterostructures was studied by a variety of characterization methods, in order to evaluate its characteristics and quality. We explored means for control over particle size, crystallinity, deposition location on the CdS rod, and composition. Illumination time and excitation wavelength were found to be key parameters for such control. The influence of different growth conditions and the characterization of these heterostructures are described alongside a detailed description of their synthesis. Of significance is the fact that the addition of iridium oxide afforded the rods astounding photochemical stability under prolonged illumination in pure water (alleviating the requirement for hole scavengers).

A protocol for the photochemical oxidative growth of small crystalline iridium oxide nanoparticles on the surface of CdSe@CdS seeded rod nanoparticles is presented.

We demonstrate a procedure for the photochemical oxidative growth of iridium oxide catalysts on the surface of seeded cadmium selenide-cadmium sulfide ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

On-line Analysis of Nitrogen Containing Compounds in Complex Hydrocarbon Matrixes

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The composition of heavy crude oils and shale oils varies substantially depending on the origin of the mixture. In particular they contain an increased amount of nitrogen containing compounds compared to the conventionally used sweet crude oils. As nitrogen compounds have an influence on the operation of thermal processes occurring in coker units and steam crackers, and as some species are considered as environmentally hazardous, a detailed analysis of the reactions involving nitrogen containing compounds under pyrolysis conditions provides valuable information. Therefore a novel method has been developed and validated with a feedstock containing a high nitrogen content, i.e., a shale oil. First, the feed was characterized offline by comprehensive two-dimensional gas chromatography (GC &#215; GC) coupled with a nitrogen chemiluminescence detector (NCD). In a second step the on-line analysis method was developed and tested on a steam cracking pilot plant by feeding pyridine dissolved in heptane. The former being a representative compound for one of the most abundant classes of compounds present in shale oil. The composition of the reactor effluent was determined via an in-house developed automated sampling system followed by immediate injection of the sample on a GC &#215; GC coupled with a time-of-flight mass spectrometer (TOF-MS), flame ionization detector (FID) and NCD. A novel method for quantitative analysis of nitrogen containing compounds using NCD and 2-chloropyridine as an internal standard has been developed and demonstrated.

A method combining comprehensive two-dimensional gas chromatography with nitrogen chemiluminescence detection has been developed and applied to on-line analysis of nitrogen containing compounds in a complex hydrocarbon matrix.

The shift to heavy crude oils and the use of alternative fossil resources such as shale oil are a challenge for the petrochemical industry. The ...

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

Molten-Salt Synthesis of Complex Metal Oxide Nanoparticles

The development of feasible synthesis methods is critical for the successful exploration of novel properties and potential applications of nanomaterials. Here, we introduce the molten-salt synthesis (MSS) method for making metal oxide nanomaterials. Advantages over other methods include its simplicity, greenness, reliability, scalability, and generalizability. Using pyrochlore lanthanum hafnium oxide (La2Hf2O7) as a representative, we describe the MSS protocol for the successful synthesis of complex metal oxide nanoparticles (NPs). Furthermore, this method has the unique ability to produce NPs with different material features by changing various synthesis parameters such as pH, temperature, duration, and post-annealing. By fine-tuning these parameters, we are able to synthesize highly uniform, non-agglomerated, and highly crystalline NPs. As a specific example, we vary the particle size of the La2Hf2O7&#160;NPs by changing the concentration of the ammonium hydroxide solution used in the MSS process, which allows us to further explore the effect of particle size on various properties. It is expected that the MSS method will become a more popular synthesis method for nanomaterials and more widely employed in the nanoscience and nanotechnology community in the upcoming years.

Here, we demonstrate a unique, relatively low-temperature, molten-salt synthesis method for preparing uniform complex metal oxide lanthanum hafnate nanoparticles.

The development of feasible synthesis methods is critical for the successful exploration of novel properties and potential applications of nanomaterials. ...

Extraction of Lignin with High &#946;-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the emerging elegant procedures to obtain specific aromatic products rely on the lignin substrate having a high content of the readily cleavable &#946;-O-4 linkage as present in the native lignin structure. This provides a miss-match with typical technical lignins that are highly degraded and therefore are low in &#946;-O-4 linkages. Therefore, the extraction yields, and the quality of the obtained lignin are of utmost importance to access new lignin valorization pathways. In this manuscript, a simple protocol is presented to obtain lignins with high &#946;-O-4 content by relatively mild ethanol extraction that can be applied to different lignocellulose sources. Furthermore, analysis procedures to determine the quality of the lignins are presented together with a depolymerization protocol that yields specific phenolic 2-arylmethyl-1,3-dioxolanes, which can be used to evaluate the obtained lignins. The presented results demonstrate the link between lignin quality and potential for the lignins to be depolymerized into specific monomeric aromatic chemicals. Overall, the extraction and depolymerization demonstrates a trade-off between the lignin extraction yield and the retention of the native aryl-ether structure and thus the potential of the lignin to be used as the substrate for the production of chemicals for higher-value applications.

Extraction of Lignin with High &amp;#946;-O-4 Content by Mild Ethanol Extraction and Its Effect on the Depolymerization Yield

Here, we present a protocol to perform ethanol extraction of lignin from several biomass sources. The effect of the extraction conditions on the lignin yield and &#946;-O-4 content are presented. Selective depolymerization is performed on the obtained lignins to obtain high aromatic monomer products.

Lignin valorization strategies are a key factor for achieving more economically competitive biorefineries based on lignocellulosic biomass. Most of the ...

Analysis of Complex Molecules and Their Reactions on Surfaces by Means of Cluster-Induced Desorption/Ionization Mass Spectrometry

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass spectrometry (MS) of complex molecules and their reactions on surfaces. DINeC is based on a beam of SO2 clusters impacting on the sample surface at low cluster energy. During cluster-surface impact, some of the surface molecules are desorbed and ionized via dissolvation in the impacting cluster; as a result of this dissolvation-mediated desorption mechanism, low cluster energy is sufficient and the desorption process is extremely soft. Both surface adsorbates and molecules of which the surface is composed of can be analyzed. Clear and fragmentation-free spectra from complex molecules such as peptides and proteins are obtained. DINeC does not require any special sample preparation, in particular no matrix has to be applied. The method yields quantitative information on the composition of the samples; molecules at a surface coverage as low as 0.1 % of a monolayer can be detected. Surface reactions such as H/D exchange or thermal decomposition can be observed in real-time and the kinetics of the reactions can be deduced. Using a pulsed nozzle for cluster beam generation, DINeC can be efficiently combined with ion trap mass spectrometry. The matrix-free and soft nature of the DINeC process in combination with the MSn capabilities of the ion trap allows for very detailed and unambiguous analysis of the chemical composition of complex organic samples and organic adsorbates on surfaces.

Neutral SO2 clusters of low kinetic energy (&#60; 0.8 eV/constituent) are used to desorb complex surface molecules such as peptides or lipids for further analysis by means of mass spectrometry using an ion trap mass spectrometer. No special sample preparation is required, and real-time observation of reactions is possible.

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass ...

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described.
Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 (MNP@SiO2) using the St&#246;ber process followed by functionalization with APTES (MNP@SiO2@NH2). Then, feroxamine was conjugated with the MNP@SiO2@NH2 by carbodiimide coupling to give MNP@SiO2@NH2@Fa. The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.

This work describes protocols for the preparation of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine using a succinyl moiety as a linker. A deep structural characterization description and a capture bacteria assay using Y. enterocolitica for all the intermediate nanoparticles and the final conjugate are also described in detail.