Novel Iodinated Dendritic Nanoparticles for Computed Tomography

Computed tomography (CT) is a reliable and widely available imaging method with high spatial resolution. The presently available CT imaging agents are...
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Novel Iodinated Dendritic Nanoparticles for Computed Tomography (CT) Imaging

2002 Vol. 2, No. 6 595-599

Alexander T. Yordanov,*,† Adriana L. Lodder,§ Eric K. Woller,| Mary J. Cloninger,| Nicholas Patronas,‡ Diane Milenic,† and Martin W. Brechbiel† Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, Center for Cancer Research, Department of Diagnostic Radiology, Clinical Center, National Institutes of Health, Bethesda, Maryland 20892, Wyatt Technology Corporation, 30 South La Patera Lane, B-7, Santa Barbara, California 93117, and Department of Chemistry and Biochemistry, Montana State UniVersity, Bozeman, Montana 59717 Received March 20, 2002

ABSTRACT The design, synthesis, and characterization of novel water-soluble iodinated dendritic nanoparticles, G-4-(DMAA-IPA)37, is reported. They consist of a generation 4 Starburst PAMAM dendrimer core and 3-N-[(N′,N′-dimethylaminoacetyl)amino]-r-ethyl-2,4,6-triiodobenzenepropanoic acid (DMAA-IPA) molecules covalently attached to the surface. These nanoparticles have an average molecular weight of 37,000 g/mol, a hydrodynamic radius of 2.4 nm and high iodine content (33.06%), while retaining their overall charge and water solubility. Thus, G-4-(DMAAIPA)37 is the first representative of a novel class of dendritic nanoparticles for computed tomography (CT) imaging.

Computed tomography (CT) is a reliable and widely available imaging method with high spatial resolution. The presently available CT imaging agents are nonspecific compounds, mostly iodinated ionic or nonionic small molecules1-3 with distribution primarily in the extracellular space. For angiographic purposes (high contrast in the cardio-vascular system) imaging is only possible on a time scale which is limited to minutes or even seconds because the low molecular weight CT agents clear very rapidly from the human body. If the peak time of the CT agent concentration in the blood is missed, a second inconvenient and potentially hazardous injection of a high volume of contrast material into the patient is necessary. The objective of extensive research efforts have therefore been the development of macromolecular CT agents which would result both in higher and in longer-lived vascular concentrations. So far, there have been relatively few reports of macromolecular, water-soluble iodinated CT agents, all of which are based upon linear synthetic polymers or functionalized starches.4-8 The structures and sizes of these carriers are presently optimized, but so far no compound has reached the status of clinical applications.9 Possible hurdles to overcome are synthetic problems such as drug uniformity, † Radioimmune and Inorganic Chemistry Section, Radiation Oncology Branch, Center for Cancer Research. ‡ Department of Diagnostic Radiology, Clinical Center, National Institutes of Health. § Wyatt Technology Corporation. | Department of Chemistry and Biochemistry, Montana State University.

10.1021/nl020246x CCC: $22.00 Published on Web 05/03/2002

© 2002 American Chemical Society

reproducible production of pure compounds and analytical issues, such as adequate characterization and demonstrating integrity. Starburst PAMAM dendrimers are commercially available synthetic spherical covalent nanoparticles composed of an ethylenediamine initiator core, repeating polyamidoamino units, and primary amino groups on the surface. They can be produced in successive “generations”, each with a specified and defined size, molecular weight, and number of terminal amino groups.10-14 Because of their well-defined characteristics and lack of immunogenicity, PAMAMs are finding utility in a variety of applications, many of which are biological in nature.15-19 Dendrimers are currently under investigation as potential polymeric carriers of contrast agents for magnetic resonance imaging (MRI)20-22 and as free radical life supporters for electron paramagnetic resonance (EPR) imaging.23 Herein, we report the synthesis and characterization of the first fully characterized water-soluble iodinated dendritic nanoparticle, G-4-(DMAA-IPA)37 (5, Scheme 1). It consists of a Starburst PAMAM Generation 4 (G-4) dendrimer that has on its surface thirty-seven (37) 3-N-[(N′,N′-dimethylaminoacetyl)amino]-R-ethyl-2,4,6-triiodobenzenepropanoic acid (DMAA-IPA) molecules covalently linked by an amide bond. Thus, the total number of iodine atoms per dendrimer unit is 111. DMAA-IPA was specifically designed so that the total positive charge (and the solubility) of the dendrimer are not affected after the attachment of the iodine-containing

Scheme 1.

Synthesis of Iodinated Nanoparticles G-4-(DMAA-IPA)37 (5).

moieties to its surface. The structure of this macromolecular conjugate as well as its purity and uniformity were confirmed by three independent methodsselemental analysis, matrixassisted laser desorption ionization time-of-flight (MALDITOF) mass spectroscopy,24 and multi-angle light scattering (MALS) analysis following fractionation by size-exclusion chromatography (SEC).25 Iodopanoic acid 1 (IPA) was purchased from TCI America (Portland, OR) and converted to 3-[(chloroacetyl)amino]-Rethyl-2,4,6-triiodobenzenepropanoic acid 2 as described elsewhere (Scheme 1).26 This compound was treated with dimethylammonium chloride in the presence of triethylamine (TEA) to give 3-N-[(N′,N′-dimethylaminoacetyl)amino]-Rethyl-2,4,6-triiodobenzenepropanoic acid 3. The watersoluble amino acid was isolated with a Waters Delta Prep 3000 Preparative Chromatography System (Milford, MA) using a water/acetonitrile gradient where both mobile phases contained 0.1% trifluoroacetic acid (TFA). The amino acid was subsequently converted to its N-hydroxysuccinimidyl active ester 4. The parent G-4 dendrimer (Aldrich Chemical Co., Milwaukee, WI) was reacted with an 100% excess of N-succinimidyl 3-N-[(N′,N′-dimethylaminoacetyl)amino]-Rethyl-2,4,6-triiodobenzenepropanoate 4 for 48 h at 80° C in DMSO in the presence of TEA. The latter was removed by evaporation and the solution diluted with 10% aqueous acetic acid and methanol. The unreacted active ester 4 and N-hydroxysuccinimide were removed by ultrafiltration with deionized water using an Amicon stirred cell (Millipore, Bedford, MA) fitted with a 10 K membrane. Occasional precipitation of a solid material in the cell occurred. In such cases, the precipitate was brought back into solution by the addition of sufficient portions of methanol, after which the filtration was continued until no more precipitation occurred upon addition of water. Then 1% aqueous NaCl was added 596

and the filtration was continued until the inside solution tested negative for Cl- ion (0.1 M AgNO3). The resulting solute was lyophilized to obtain the desired iodinated nanoparticles G-4-(DMAA-IPA)37 (5) as light brownish fluffy powder. We have characterized our iodine-labeled-G-4 Starburst PAMAM dendrimer by three independent methods. Because iodine is the element accounting for the clinically important X-ray absorbing properties of this material, its elemental content is a major characteristic of any candidate for CT imaging purposes. The elemental analysis (Galbraith Laboratories Inc., Knoxville, TN) of our G-4-(DMAA-IPA)37 (5) gave the following results: C, 35.07%; H, 5.09%; N, 9.35%; I, 33.06%. Its high iodine content (almost one-third of its weight) fulfills the most important prerequisite for a potential CT imaging agent. To determine the number of 3-N-[(N′,N′-dimethylaminoacetyl)amino]-R-ethyl-2,4,6-triiodobenzenepropanoic acid molecules on the surface in G-4-(DMAA-IPA)37 (5), we compared its MALDI-TOF spectra to that of the parent G-4 dendrimer. MALDI mass spectra were acquired using a Bruker Biflex-III time-of-flight mass spectrometer (Billerica, MA). Positive ion mass spectra were acquired in linear mode, and the ions were generated using a nitrogen laser (337 nm) pulsed at 3 Hz with a pulse width of 3 ns.27 Ions were accelerated at 19 000-20 000 V and amplified using a discrete dynode multiplier. Spectra (100 to 200) were summed into a LeCroy LSA1000 high-speed signal digitizer. All data processing was performed using Bruker XMass/ XTOF V 5.0.2. Trans-3-indoleacrylic acid matrix was used with a matrix-analyte ratio of 3000:1 or 1000:1. DMF was used as the solvent for all solutions except the standards. Horse heart myoglobin (MW 16,952 g/mol) and bovine serum albumin (BSA, MW 66,431 g/mol) were used as external standards. An aliquot corresponding to 12-15 pmol of analyte was deposited on the laser target. The parent G-4 PAMAM dendrimer has a MW average of 13,450 g/mol. The MALDI-TOF spectrum of G-4(DMAA-IPA)37 is shown in Figure 1. Similar distribution patterns of dendrimers have been described previously.28 The iodinated dendritic nanoparticles have a weight-average molar mass (Mw) 37,000 g/mol; number average molar mass (Mn) 36,657 g/mol; polydispersity (Mw/Mn) 1.0121. On the basis of the molecular weight increase, we calculated that there are thirty-seven (37) 3-N-[(N′,N′-dimethylaminoacetyl)amino]-R-ethyl-2,4,6-triiodobenzenepropanoic acid (3) residues on the surface of the dendrimer. Although there are as many as 64 primary amino groups on the G-4 surface, we were unable to conjugate more than thirty-seven (37) DMAA-IPA molecules to the dendrimer surface even under the harsh labeling conditions described above. No doubt this is a result of the accumulated steric hindrance of the bulky DMAA-IPA molecules on the dendrimer surface preventing access to the unreacted primary amino groups. The MALDI-TOF spectrum also shows an apparently large percentage of dimer formation. While noncovalent di-, tri-, and polymerization is generally common under the conditions of the MALDI-TOF spectroscopy, this particular sample showed more dimer than usually seen under these conditions. Nano Lett., Vol. 2, No. 6, 2002

Figure 1. MALDI-TOF spectrum of G-4-(DMAA-IPA)37, 5.

This observation was not surprising when considering the lipophilic nature of the G-4-(DMAA-IPA)37 dendrimer surface. However, we were curious as to whether it was possible that dimeric polymers were somehow covalently formed under our synthetic conditions (unlikely as this might seem) because the variation of the dendrimer concentration from 3000:1 to 1000:1 did not cause any major difference in the MALDI-TOF spectrum with exception to overall intensity. The presence of covalent oligomers in an imaging agent would be an undesirable characteristic because even a small proportion present could potentially obstruct narrow blood capillaries in tissues, result in precipitable masses in vivo, and thus be a source of toxicity. To address this issue, a sample of our G-4-(DMAA-IPA)37 conjugate was analyzed by MALS following fractionation by SEC. A Waters Alliance 2690 HPLC equipped with a TSK G3000PWxl methacrylate beads size-exclusion column was used along with a Wyatt Technology miniDAWN combined with Wyatt Technology QELS (quasi-elastic light scattering) detector (Santa Barbara, CA). The Wyatt Technology Optilab DSP, a differential refractometer (DRI detector) was used as concentration detector. The data were collected and analyzed by Wyatt Technology ASTRA for Nano Lett., Vol. 2, No. 6, 2002

Windows software version 4.81. The mobile phase was 0.5 M aqueous acetic acid/methanol (1:1) and the flow rate was 0.5 mL/min. 1 mg of sample was loaded on the column (sample concentration: 10 mg/mL, injection volume: 100 µL). The chromatograms obtained from SEC of the G-4(DMAA-IPA)37 sample are depicted in Figure 2. The total inclusion volume of the column is at approximately 20 min from where the DRI signals were observed from salts and air in the sample. The sample eluted after the total inclusion volume indicating the elution of the sample is based upon nonsize exclusion effects. Under the same conditions, the protein BSA eluted around 25 min. This suggests that column interaction is stronger for the dendrimer than for BSA. Because the dendrimer consists largely of peptide-like bonds, the specific refractive index increment (dn/dc) value that was measured for the protein BSA, 0.185 mL/g, was also applied for the dendrimer sample. The molar masses of the molecules were calculated from each data slice of the chromatogram and are plotted against the elution time in Figure 3. The chromatogram of the DRI detector is superimposed. The results are as follows: weight-average molar mass (Mw) 42,000 g/mol; number-average molar mass (Mn) 41,000 g/mol; polydispersity (Mw/Mn) 1.03. QELS was also 597

Figure 2. Chromatograms obtained by SEC-MALS of the G-4-(DMAA-IPA)37 dendrimer sample with signals from the 90° LS (top) and DRI (bottom) detectors.

Figure 3. Plot of the molar mass versus elution time (bold line) superimposed with the signal from the DRI detector (thin line) obtained from SEC-MALS of the G-4-(DMAA-IPA)37 dendrimer sample.

performed in order to determine the hydrodynamic radius (Rh) of the dendrimer molecule. The data were collected every 10 s, and from the correlation function, the radius was determined to be 2.4 nm. The results for Rh of the dendrimer sample, superimposed with the signal from DRI detector, are presented in Figure 4. It is interesting to note that the hydrodynamic radius is much smaller compared to BSA, a globular protein with a molar mass of 66,431 g/mol and Rh of 3.4 nm. This indicates that the G-4-(DMAA-IPA)37 dendrimer has a very dense structure which agrees with its high iodine content (33.06%). The molar mass distribution plot shows a plateau (Figure 3), an indication that the sample is quite monodisperse, since molar mass is constant for the majority of the eluted peak. The molar mass value obtained by the MALS determination is slightly higher than that obtained from the MALDI-TOF spectrum. There are two possible explanations for this phenomenon. One reason for possible overestimation of molar mass by MALS could be that the dn/dc value used (0.185 mL/g as measured for BSA) is too low. Because the 598

Figure 4. Results for the hydrodymanic radius (Rh) (dots) of the G-4-(DMAA-IPA)37 dendrimer sample calculated from the SECQELS data, superimposed with the DRI signal (line).

dendrimer is highly charged the actual dn/dc value may be higher. A higher dn/dc value would result in a lower molar mass result. There are no previous reports in the literature of highly charged water-soluble dendrimer molar mass determinations by MALS. A second reason for a higher molar mass obtained by MALS can be the inclusion of slowly exchanging solvent molecules and counterions within the dendrimer in solution. If one accepts a molar mass of 42,000 g/mol and 37 terminal DMAA-IPA residues (as calculated from the MALDI-TOF analysis) then the theoretical iodine content is 33.0%, which is exactly what was found by the elemental analysis (33.06%). Even after lyophilization solvent molecules and counterions stay trapped in the dendrimer sample. Thus, the elemental analysis data should correspond to the hydrated state of the dendrimer in the MALS analysis rather than to the one in the MALDI-TOF spectrum which does not include any small molecules. Also, as seen from Figure 3, there is no indication of the presence of any covalent dimers in the sample because those would appear as separate peaks with a longer retention time and would disturb the molar mass distribution plateau. It is Nano Lett., Vol. 2, No. 6, 2002

necessary to underline that the elution of this sample was based not only on size-exclusion but on column material interaction as well, in which case larger molecules are expected to elute later than smaller ones because they have more surface to interact with the column material. In summary, novel water-soluble iodinated dendritic nanoparticles have been synthesized and characterized by three independent methods. Their uniformity has been established and their composition and molecular weight have been determined by elemental analysis, MALDI-TOF spectroscopy and MALS analysis. The two numbers for the molecular weight thereby obtained are in sufficiently good agreement. Our study has shown that the iodine-labeled nanoparticle G-4-(DMAA-IPA)37 is a potential candidate for a CT imaging agent. Currently, murine model studies are under way for further biological evaluation, and those results will be reported in the appropriate venue. This work also demonstrates the applicability of the different physical methods and their combined application for a full determination of the molecular weight, size, composition, and integrity of water-soluble dendritic nanoparticles. References (1) Cohan, R. H.; Ellis, J. H. Urol. Clin. N. Am. 1997, 24(3), 471. (2) Krause, W. AdV. Drug DeliVer. ReV. 1999, 37(1-3), 159-173. (3) Lee, A. G.; Hayman, L. A.; Ross, A. W. SurV. Ophthalmol. 2000, 45(3), 237-253. (4) Trubetskoy, V. S.; Gazelle, G. S.; Wolf, G. L.; Torchilin, V. P. J. Drug Target. 1997, 4(6), 381-388. (5) Sachse, A.; Leike, J. U.; Schneider, T.; Wagner, S. E.; Roebling, G. L.; Krause, W.; Brandl, M. InVest. Radiol. 1997, 32(1), 44-50. (6) Bogdanov, A. A., Jr.; Weissleder, R.; Brady, T. J. AdV. Drug DeliVer. ReV. 1995, 16(2-3), 335-348. (7) Lautrou, J.; Paris, D.; Schaefer, M.; Meyer, D.; Chambon, C.; Doucet, D. InVest. Radiol. 1990, 25(Suppl. 1), S109-S110. (8) Sako, M.; Watanabe, H.; Okuda, K.; Shimizu, T.; Hase, M.; Hirota, S.; Kono, M.; Sakamoto, K. Nippon Igaku Hoshasen Gakkai Zasshi 1987, 47(11), 1472-1477. (9) Krause, W.; Hackmann-Schlichter, N.; Maier, F. K.; Muller, R. Top. Curr. Chem. 2000, 210, 261-308.

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