Preparation and In Vitro Characterization of Dendrimer-based Contrast Agents for Magnetic Resonance Imaging

Summary

This protocol describes the preparation and characterization of a dendrimeric magnetic resonance imaging (MRI) contrast agent that carries cyclen-based macrocyclic chelates coordinating paramagnetic gadolinium ions. In a series of MRI experiments in vitro, this agent produced an amplified MRI signal when compared to the commercially available monomeric analogue.

Abstract

Paramagnetic complexes of gadolinium(III) with acyclic or macrocyclic chelates are the most commonly used contrast agents (CAs) for magnetic resonance imaging (MRI). Their purpose is to enhance the relaxation rate of water protons in tissue, thus increasing the MR image contrast and the specificity of the MRI measurements. Current clinically approved contrast agents are low molecular weight molecules that are rapidly cleared from the body. The use of dendrimers as carriers of paramagnetic chelators can play an important role in the future development of more efficient MRI contrast agents. Specifically, the increase in local concentration of the paramagnetic species results in a higher signal contrast. Furthermore, this CA provides a longer tissue retention time due to its high molecular weight and size. Here, we demonstrate a convenient procedure for the preparation of macromolecular MRI contrast agents based on poly(amidoamine) (PAMAM) dendrimers with monomacrocyclic DOTA-type chelators (DOTA – 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate). The chelating unit was appended by exploiting the reactivity of the isothiocyanate (NCS) group towards the amine surface groups of the PAMAM dendrimer to form thiourea bridges. Dendrimeric products were purified and analyzed by means of nuclear magnetic resonance spectroscopy, mass spectrometry, and elemental analysis. Finally, high resolution MR images were recorded and the signal contrasts obtained from the prepared dendrimeric and the commercially available monomeric agents were compared.

Introduction

Magnetic resonance imaging (MRI) is a powerful and non-ionizing imaging technique widely used in biomedical research and clinical diagnostics due to its noninvasive nature and excellent intrinsic soft-tissue contrast. The most commonly used MRI methods utilize the signal obtained from water protons, providing high-resolution images and detailed information within the tissues based on differences in the density of the water signals. The signal intensity and the specificity of the MRI experiments can be further improved using contrast agents (CAs). These are paramagnetic or superparamagnetic species that affect the longitudinal (T₁) and transverse (T₂) relaxation times, respectively^1,2.

Complexes of the lanthanide ion gadolinium with polyamino polycarboxylic acid ligands are the most commonly used T₁ CAs. Gadolinium(III) shortens the T₁ relaxation time of water protons, thus increasing the signal contrast in MRI experiments³. However, ionic gadolinium is toxic; its size approximates that of calcium(II), and it seriously affects calcium-assisted signaling in cells. Therefore, acyclic and macrocyclic chelates are employed to neutralize this toxicity. Various multidentate ligands have been developed so far, resulting in gadolinium(III) complexes with high thermodynamic stability and kinetic inertness¹. Those based on the 12-membered azamacrocycle cyclen, in particular its tetracarboxylic derivative DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate) are the most investigated and applied complexes of this CA class.

Nevertheless, GdDOTA-type CAs are low molecular weight systems, displaying certain disadvantages such as low contrast efficiency and fast renal excretion. Macromolecular and multivalent CAs may be a good solution to these problems⁴. Since CA biodistribution is mainly determined by their size, macromolecular CAs display much longer retention times within tissues. Equally important, the multivalency of these agents results in an increased local concentration of the monomeric MR probe (e.g., GdDOTA complex), substantially improving the acquired MR signal and the measurement quality.

Dendrimers are amongst the most preferred scaffolds for the preparation of multivalent CAs for MRI^4,5. These highly branched macromolecules with well-defined sizes are prone to various coupling reactions on their surface. In this work, we report the preparation, purification, and characterization of a dendrimeric CA for MRI consisting of a generation 4 (G4) poly(amidoamine) (PAMAM) dendrimer coupled to GdDOTA-like chelates (DCA). We describe the synthesis of the reactive DOTA derivative and its coupling to the PAMAM dendrimer. Upon complexation with Gd(III), the standard physicochemical characterization procedure of DCA was performed. Finally, MRI experiments were performed to demonstrate the ability of DCA to produce MR images with a stronger contrast than those obtained from low molecular weight CAs.

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Protocol

1. Preparation of DCA

1. Synthesis of the monomeric unit 4⁶.
   1. Synthesis of 4-(4-nitrophenyl)-2-(4,7,10-tris-tert-butoxycarbonylmethyl-1,4,7,10-tetraazacyclododec-1-yl)butyric acid tert-butyl ester (2).
      1. Dissolve (4,7-bis-tert-butoxycarbonylmethyl-1,4,7,10-tetraaza-cyclododec-1-yl)-acetic acid tert-butyl ester 1 (1.00 g, 1.94 mmol) in N,N-dimethylformamide (DMF, 5 ml), add potassium carbonate (0.67 g, 4.86 mmol, 2.5 equiv.) and stir the mixture at room temperature for 45 min.
         NOTE: Macrocycle 1 was prepared from cyclen and tert-butyl bromoacetate according to the previously published procedure⁷.
      2. Add tert-butyl-2-bromo-4-(4-nitrophenyl)butanoate (0.87 g, 2.53 mmol, 1.3 equiv.) portionwise over 1 hr. Continue stirring the mixture under the same reaction conditions for the following 18 hr.
         Note: Tert-butyl-2-bromo-4-(4-nitrophenyl)butanoate was prepared from 4-(4-nitrophenyl)-butyric acid, thionyl chloride, and bromine according to the previously published procedure⁸.
      3. Remove DMF by means of bulb-to-bulb vacuum distillation at 40-60 °C⁹.
      4. Purify the residue by column chromatography (silica gel, 7% methanol/dichloromethane) to obtain product 2 as a brown amorphous solid (1.09 g, 72%)¹⁰.
   2. Synthesis of 4-(4-aminophenyl)-2-(4,7,10-tris-tert-butoxycarbonylmethyl-1,4,7,10-tetraazacyclododec-1-yl)butyric acid tert-butyl ester (3).
      1. Dissolve the nitrobenzene derivative 2 (1.00 g, 1.28 mmol) in ethanol (10 ml) and 7 N ammonia solution in methanol (150 µl). Add palladium on activated carbon as a catalyst (Pd/C, 150 mg, 15 wt %) to the solution.
      2. Shake the heterogeneous mixture for 16 hr under a hydrogen atmosphere (2.5 bar) in the Parr hydrogenator apparatus.
      3. Prepare a cake of diatomaceous earth by suspending it in ethanol and filtering the suspension through a sintered glass funnel. Pour the suspension from 1.1.2.2 over the prepared cake to remove the Pd/C catalyst by filtration.
      4. Remove the solvent by gentle distillation on a rotary evaporator (water bath temperature ~40 °C) to obtain compound 3 as a brown amorphous solid (0.91 g, 95%).
   3. Synthesis of 4-(4-isothiocyanatophenyl)-2-(4,7,10-tris-tert-butoxycarbonylmethyl-1,4,7,10- tetraazacyclododec-1-yl)butyric acid tert-butyl ester (4).
      1. Add thiophosgene (0.124 ml, 1.58 mmol, 1.3 equiv.) to a mixture of 3 (0.91 g, 1.22 mmol) and triethylamine (0.685 ml, 4.87 mmol, 4 equiv.) in dichloromethane (15 ml).
      2. Vigorously stir the reaction mixture with a magnetic stirrer at room temperature for 16 hr.
      3. Remove the solvent by gentle distillation on a rotary evaporator (water bath temperature ~40 °C), and then purify the crude product by column chromatography (silica gel, 5% methanol/dichloromethane) to obtain the product 4 as a light brown amorphous solid (0.51 g, 53%).
2. Synthesis of the dendrimer DCA.
   1. Synthesis of the dendrimer 5.
      1. Take G4-PAMAM dendrimer (667 mg, 10% dendrimer solution in methanol, 4.67 µmol), evaporate the methanol by gentle distillation on a rotary evaporator (water bath temperature ~40 °C), and dissolve the residue in DMF (4 ml).
      2. Add triethylamine (0.105 ml, 0.75 mmol, 160 equiv.), stir for 45 min at 60 °C, and add isothiocyanate 4 (354 mg, 0.45 mmol, 1.5 equiv. relative to the amino surface groups of the dendrimer) portionwise over 1 hr.
      3. Stir the reaction mixture with a magnetic stirrer at 45 °C for 48 hr.
      4. Remove the solvent by means of bulb-to-bulb vacuum distillation at 40-60 °C.
      5. Purify the residue by size-exclusion chromatography using a lipophilic gel filtration medium and methanol as the eluent. To pack the column, swell the filtration media in methanol for at least 3 hr at room temperature (>4 ml of methanol per 1 g of powder) without applying pressure. Perform gravity separation by collecting 1 ml fractions.
      6. Analyze the collected fractions with thin-layer chromatography (TLC). Develop the TLC plate in 15% methanol/dichloromethane (only the most polar spot located on the base line is derived from dendrimeric product). Evaporate the collected fractions by gentle distillation on a rotary evaporator (water bath temperature ~40 °C) to obtain product 5 (270 mg, 91%).
   2. Synthesis of the dendrimer 6.
      1. Dissolve the protected dendrimeric chelator 5 (270 mg, 4.23 µmol) in formic acid (5 ml) and stir the mixture at 60 °C for 24 hr.
      2. Evaporate the formic acid by distillation on a rotary evaporator (~15 mbar pressure, water bath temperature ~40 °C) and freeze-dry the product to give 6 (pressure ~0.2 mbar)⁹.
   3. Synthesis of the dendrimeric contrast agent (DCA)
      1. Dissolve the dendrimeric chelator 6 (4.35 µmol) in water and adjust the pH to 7.0 with 0.1 M sodium hydroxide.
      2. Dissolve GdCl₃·6H₂O (113 mg, 304 µmol) in water (1 ml) and add it dropwise to the solution of chelator 6 over a period of 4 hr; maintain the pH at 7.0 with aqueous sodium hydroxide solution (0.05 M) by measuring pH with a pH meter.
      3. Stir the mixture with a magnetic stirrer at room temperature for 24 hr.
      4. Add ethylenediaminetetraacetic acid (EDTA, 158 mg, 426 µmol) to the solution portionwise over 4 hr to remove the excess of Gd(III) while maintaining the pH at 7.0 with aqueous sodium hydroxide solution (0.05 M). Stir the mixture at room temperature for 24 hr.
      5. Perform size-exclusion chromatography to remove the majority of GdEDTA and the excess of EDTA. Use a hydrophilic gel filtration medium swollen in water to pack the column. Reduce the mixture to a suitable volume and load the column. Elute the column with deionized water without applying pressure.
      6. Centrifuge the sample using a 3 kDa centrifugal filter unit for 30 min at centrifugal force 1,800 x g to remove the residues of GdEDTA and EDTA. Repeat this step (around five times) until the filtrate shows the absence of EDTA and GdEDTA. Transfer the sample into a flask, evaporate it, and then freeze-dry the solvent to obtain an off-white product as the final DCA (186 mg, 71%).
         NOTE: Check absence of EDTA and GdEDTA by means of ESI-MS.
      7. Confirm the absence of Gd(III) as a free ion using the xylenol orange test. Dissolve the filtrate (0.5 ml) in an acetate buffer solution (pH 5.8). Add a few drops of a xylenol orange solution and track the color change (yellow or violet color indicates the absence or presence of free Gd(III) ions in solution, respectively)¹¹.

2. In Vitro Characterization of Dendrimeric Products

1. Estimation of the number of macrocyclic DOTA-units coupled to the PAMAM dendrimer (loading of the dendrimer with DOTA-like macrocycles)
   1. Estimation with ¹H NMR (NMR — nuclear magnetic resonance spectroscopy).
      NOTE: This procedure is possible on dendrimers 5 and 6, but not on DCA.
      1. Record the ¹H NMR spectrum¹².
      2. Integrate the aromatic region and the two separate aliphatic regions (1. signals of the aliphatic dendrimer and macrocyclic protons; 2. signals of the t-Bu groups) or just an aliphatic region for dendrimers 5 and 6, respectively.
         Note: There is no separate signal in the aliphatic region originated from the t-Bu groups in dendrimer 6 since they have been hydrolyzed.
      3. Use Eq. 1 or Eq. 2 to estimate the number of macrocyclic units (n), where R = the ratio of integrals (aliphatic/aromatic in Eq. 1 or aliphatic-dendrimer/aliphatic-t-Bu in Eq. 2), H_dend = the number of protons in dendrimer, H_Ar = the number of aromatic protons, H_tBu= the number of protons in t-Bu groups, and H_mac = the number of protons in one macrocycle.
         Note: Either Eq. 1 or Eq. 2 can be used for dendrimer 5, while only Eq. 1 can be used for dendrimer 6. Since exchangeable protons (on amines, amides, thioureas, or carboxylates) are typically being replaced with deuterium, they were not assumed in the calculations. Here, H_dend = 1,128 (for 5) or 1,000 (for 6), H_Ar = 4, and H_mac = 27 were used.
         (1)
         (2)
   2. Estimation from elemental analysis by using the ratio of nitrogen to sulphur.
      1. Perform the elemental analysis on the solid dendrimeric sample (DCA in this work).
      2. Use Eq. 3 to estimate the number of macrocyclic units (n), where R = the ratio of determined %N and %S, N_dend or S_dend = the number of nitrogen or sulphur atoms in the dendrimer, and N_mac or S_mac = the number of nitrogen or sulphur atoms in one macrocyclic unit.
         Note: The factor 2.29 is obtained from the ratio in atomic masses of sulphur and nitrogen. In this work, N_dend = 250, S_dend = 2, N_mac = 5, and S_mac = 1 were used.
         (3)
   3. Estimation with matrix-assisted laser desorption/ionization time of flight (MALDI-TOF).
      1. Perform the MALDI-TOF MS analysis¹³.
      2. Calculate the number of macrocyclic units (n) according to Eq. 4, where M_z = the observed mass (m/z), z = the charge of the species, M_dend = the mass of the dendrimeric part, and M_mac = the mass of one macrocyclic unit.
         NOTE: M_dend = 14,306 and M_mac = 719 were used in this work.
         (4)
2. Determination of DCA concentration ([DCA]): Bulk magnetic susceptibility measurement (BMS)
   1. Dissolve DCA (5-10 mg) in water (360 µl) in a plastic vial tube ([DCA] ~5-10 mM).
      NOTE: [DCA] should be in the range of 5-10 mM to avoid possible overlap of t-BuOH resonances at sample concentrations >15 mM, with the resonance of water at δ = 4.7 ppm.
   2. Add 60 µl of D₂O:t-BuOH mixture (2:1 v/v) to the aqueous solution of DCA and mix the resulting solution (420 µl) using a Vortex mixer.
   3. Transfer 400 µl of the sample into an outer NMR tube and place a coaxial NMR insert tube with a t-BuOH:H₂O mixture (10:90 v/v) into the sample tube.
   4. Record the ¹H NMR spectrum and measure the frequency shift between resonance signals deriving from t-BuOH in the inner and outer NMR tubes (reference)¹².
   5. Use Eq. 5 to determine the [DCA], where T = the absolute temperature, Δχ = the recorded shift, µ_eff = the effective magnetic moment for a lanthanide ion (µ_eff = 7.94 for Gd(III)¹⁴, and s = a constant dependent upon the shape of the sample and its position in the magnetic field (0, 1/3, and 1/6 in the case of a sphere, cylinder parallel to, and cylinder perpendicular to the magnetic field, respectively).
      NOTE: The calculated value obtained for the [DCA] should be corrected to the original concentration due to the addition of the D₂O:t-BuOH solution (60 µl).
      (5)
3. Dynamic light scattering (DLS) measurements.
   1. Prepare a filtered DCA solution (0.2 µm polytetrafluorethylene/PTFE filter, 0.75 mM per Gd(III)) in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (25 mM, pH 7.4) and transfer it into a cuvette for the DLS measurement.
   2. Place the cuvette into the DLS apparatus and set the following parameters: 5 repetitions of 15 scans (1 scan = 12 sec, refractive index = 1.345, absorption = 1%) without delays in between the scans and with temperature equilibration 30 sec prior to recording.
   3. Export the acquired data and obtain the size distribution histogram by plotting population (%) as a function of size (hydrodynamic diameter).
4. Measurement of the longitudinal and transverse relaxivities.
   NOTE: A similar procedure was already described using the relaxation time analyzer¹⁵; this procedure was performed using a 300 MHz NMR spectrometer with Topspin software.
   1. Prepare a set of DCA solutions in H₂O:D₂O (500 µl, 10% D₂O in H₂O, [DCA] = 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, and 5.0 mM, [HEPES] = 25 mM) from the DCA stock sample (see section 2.2).
   2. Transfer 450 µl of solution into an NMR tube and place it into the instrument.
   3. Optimize the acquisition parameters (90° excitation pulse duration (p1), and irradiation frequency offset (O₁)) and then perform the T₁ and T₂ experiments using the inversion recovery (IR) and Car-Purcell-Meiboom-Gill (CPMG) pulse sequences, respectively.
   4. Determination of T₁ and T₂ relaxation times.
      1. Select the recorded measurement, process the 2D spectrum in the F2 dimension, and perform the interactive phase correction.
      2. Select the appropriate slice (peak with maximum intensity) in the Analysis/ T₁/T₂ relaxation window, integrate it, and export the region to the relaxation module.
      3. Select the appropriate fitting function (invrec or uxnmrt2 for IR and CPMG experiments, respectively) to obtain the T₁ or T₂ relaxation times.
   5. Repeat steps 2.4.4.2-2.4.4.4 for all the remaining [DCA] solutions.
   6. Calculate the relaxation rates (R₁ and R₂) from the obtained T₁ values (R_1,2=1/T_1,2).
   7. Plot R₁ and R₂ (sec^-1) as a function of Gd(III) concentration in mM.
   8. Determine the longitudinal and transverse relaxivities, r₁ and r₂ (mM^-1 sec^-1), from the slope of the fitted line, as defined by Eq. 6, where R_i,obs = the longitudinal (i=1) or transverse (i=2) diamagnetic relaxation rate of water in the absence of paramagnetic species and [Gd] = the concentration of Gd(III) used in the experiment.
      (6)

3. In Vitro MRI; Comparison Between DCA and GdDOTA

1. Preparation of tube phantoms
   1. Prepare aqueous solutions of DCA (4 x 350 µl) and GdDOTA (4 x 350 µl) as well as water samples (4 x 350 µl) for two sets of experiments where the concentration of the contrast agents is calculated: (3.1.1.1) per Gd(III) or (3.1.1.2) per molecule.
      1. Prepare two DCA samples and two GdDOTA samples with concentrations of 0.5 and 1.0 mM per Gd(III), respectively. Additionally, prepare two water samples (as control tubes).
      2. Prepare two DCA samples (2.5 and 5.0 mM per Gd(III) or 0.05 and 0.1 mM per dendrimeric molecule), two GdDOTA samples (0.25, 0.5 mM) and two water samples (control tubes).
         NOTE: The appropriate DCA and GdDOTA concentrations should be prepared by diluting the respective stock samples with concentrations determined via the BMS method (see section 2.2) with HEPES buffer (pH 7.4). In order to simplify the calculations, n = 50 was assumed for the average number of macrocyclic units per dendrimer molecule. Therefore, the ratio of DCA:GdDOTA was 1:5, calculated on a per molecule basis.
   2. Place the samples in 300 µl plastic vial tubes, avoiding the presence of air bubbles in the solution.
      NOTE: The size of the plastic vial tubes depends on the type and size of the radiofrequency coil used (here, an example with the volume coil is given).
   3. Insert the samples inside a syringe (60 ml volume), fill it with 1 mM GdDOTA solution, and place it in the scanner.
      NOTE: Samples were placed in the aqueous solution of GdDOTA to avoid susceptibility effects (variations in the magnetic field strength that occur near interfaces between substances of different magnetic susceptibility).
2. Parameter optimization and imaging.
   1. Use the anatomical scan (Localizer/Tripilot) to position the syringe with the samples in the isocenter of the magnet.
   2. Press the traffic light (adjustment scan) to perform adjustments for shimming (adjustment of magnetic field homogeneity) of the whole volume, the central frequency (O₁), the receiver gain (RG), and the transmit gain (TX0 and TX1).
   3. For T₁-weighted (T_1w) imaging, select the fast low angle shot (FLASH) method.
   4. Choose coronal slice for the samples placed vertically (syringe horizontally) in the scanner using the Localizer scan.
   5. Use Eq. 7 for optimization of the contrast-to-noise (CNR) acquisition parameters¹⁶, where α = the flip angle, TE = the echo time, TR = the repetition time, and T₁,_A, T_1,B = the T₁ times of sample A (T₁,_A) and sample B (T_1,B) for which the CNR should be maximized (the same is valid for T₂ times: T_2,A and T_2,B).
      NOTE: T₁ and T₂ relaxation times should be set to values obtained from the measurements of longitudinal and transverse relaxivities (section 2.4), while TE, TR, and α should be obtained from the CNR optimization calculation.
      (7)
   6. Acquire the image using the parameters obtained in the previous step (3.2.5).
   7. Calculate the signal-to-noise ratio (SNR).
      1. Load the acquired T_1w image (scan) into the Image display & processing window, and click on Define region of interest (ROI).
      2. Choose a circular ROI and draw it at the sample position and background. Subsequently, click on display to obtain the average signal amplitude (S_signal) and standard deviation of the background (S_noise).
      3. Repeat step 3.2.7.2 for the DCA, GdDOTA, and water samples.
      4. Calculate the SNR using the formula: SNR = S_signal / S_noise.
   8. Following a slightly modified procedure, perform T₂-weighted (T_2w) imaging using the rapid acquisition with relaxation enhancement (RARE) method. For optimization of the CNR acquisition parameters, use Eq. 8.
      (8)

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Results

The preparation of DCA consisted of two stages: 1) synthesis of the monomeric DOTA-type chelator (Figure 1) and 2) coupling of the chelator with the G4 PAMAM dendrimer and subsequent preparation of the dendrimeric Gd(III) complex (Figure 2). In the first stage, a cyclen-based DOTA-type chelator containing four carboxylic acids and an orthogonal group suitable for further synthetic modifications was prepared. The preparation commenced from...

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Discussion

Preparation of the dendrimeric MRI contrast agent requires appropriate selection of the monomeric unit (i.e., the chelator for Gd(III)). They reduce the toxicity of this paramagnetic ion and, to date, a wide variety of acyclic and macrocyclic chelators serve this purpose^1-3. Among these, macrocyclic DOTA-type chelators possess the highest thermodynamic stability and kinetic inertness and, hence, are the most preferred choice for the preparation of inert MRI contrast agents^1,18. Furthermore,...

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Materials

Name	Company	Catalog Number	Comments
Cyclen	CheMatech	C002
tert-Butyl bromoacetate	Alfa Aesar	A14917
N,N-Dimethylformamide	Fluka	40248
Potassium carbonate	Sigma-Aldrich	209619
4-(4-Nitrophenyl)butryic acid	Aldrich	335339
Thionyl chloride	Acros Organics	382662500	Note: Corrosive substance; toxic if inhaled
Bromine	Acros Organics	402841000	Note: causes severe skin burns, fatal if inhaled
Diethyl ether	any source
Sodium sulphate	Acros Organics	196640010
Chloroform	VWR Chemicals	22711.29
tert-Butyl 2,2,2-trichloroacetimidate	Aldrich	364789	Note: flammable substance; irritrant to skin and eyes
Boron trifluoride etherate	Acros Organics	174560250	48% BF3. Note: Flammable substance; causes skin burns, fatal if inhaled
Sodium bicarbonate	Acros Organics	424270010
Ethyl-acetate	any source		For column chromatography
n-Hexane	any source		For column chromatography
Bulb-to-bulb (Kugelrohr) distillation apparatus	Büchi		Model type: Glass oven B-585
Silicagel	Carl Roth GmbH	P090.2
Methanol	any source		For column chromatography
Dichloromethane	any source		For column chromatography
Ethanol	VWR Chemicals	20821.296
Ammonia	Acros Organics	428381000	7 N Solution in Methanol
Palladium	Aldrich	643181	15% wet
Hydrogenation apparatus PARR	PARR Instrument Company
Celite 503	Aldrich	22151
Sintered glass funnel	any source
Thiophosgen	Aldrich	115150	Note: irritrant to skin; toxic if inhaled
Triethylamine	Alfa Aesar	A12646
Dichloromethane	Acros Organics	348460010	Extra dry
Magnetic stirrer	any source
PAMAM G4 Dendrimer	Andrews ChemService	AuCS - 297	10% wt. solution in MeOH
Lipophylic Sephadex LH-20	Sigma	LH20100
Thin-layer chromatography plates	Merck Millipore	1.05554.0001
Formic acid	VWR Chemicals	20318.297
Lophylizer	any source
Gadollinium(III) chloride hexahydrate	Aldrich	G7532
Sodium hydroxide	Acros Organics	134070010
pH meter	any source
Ethylenediaminetetraacetic acid disodium salt dihydrate	Aldrich	E5134
Mass spectrometer (ESI)	Agilent		Ion trap SL 1100
Acetate buffer	any source		pH 5.8
Xylenol orange	Aldrich	52097	20 μM in acetate buffer
Hydrophylic Sephadex G-15	GE Healthcare	17-0020-01
Amicon Ultra-15 Centrifugal Filter Unit	Merck Millipore	UFC900324	Ultracel-3 membrane (MWCO 3000)
Centrifuge	any source
NMR spectrometer	Bruker		Avance III 300 MHz
Topspin	Bruker		Version 2.1
Combustion analysis instrument	EuroVector SpA		EuroEA 3000 Elemental Analyser
MALDI-ToF MS instrument	Applied Biosystems		Voyager-STR
Deuteriumoxid	Carl Roth GmbH	6672.3
tert-Butyl alcohol	Carl Roth GmbH	AE16.1
Vortex mixer	any source
Norell NMR tubes	Deutero GmbH	507-HP-7
NMR coaxial tube	Deutero GmbH	coaxialb-5-7
DLS instrument	Malvern		Zetasizer Nano ZS
0.20 μm PTFE filter	Carl Roth GmbH	KC94.1
HEPES	Fisher BioReagents	BP310
Plastic tube vials	any source
Dotarem	Guerbet		NDC 67684-2000-1
MRI scanner	Bruker		BioSpec 70/30 USR magnet (7 T). Note: potential hazards related to high magnetic fields
RF coil	Bruker		Dual frequency volume coil (RF RES 300 1H/19F 075/040 LIN/LIN TR)
Paravision (software)	Bruker		Version 5.1