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. 2012 Mar 5;9(3):374-81.
doi: 10.1021/mp2003219. Epub 2011 Sep 21.

Preparation of cystamine core dendrimer and antibody-dendrimer conjugates for MRI angiography

Kido Nwe  1 , Diane E Milenic, Geoffrey L Ray, Young-Seung Kim, Martin W Brechbiel
Affiliations

Preparation of cystamine core dendrimer and antibody-dendrimer conjugates for MRI angiography

Kido Nwe et al. Mol Pharm. .

Abstract

Herein we report the preparation along with the in vivo and in vitro MRI characterization of two generation four and five cystamine core dendrimers loaded with thirty and fifty-eight derivatized Gd-DOTA (G4SS30, G5SS58) respectively. Likewise the development and characterization of two half-dendrimers conjugated to the F(ab')(2) fragment of the monoclonal antibody (mAb) panitumumab functionalized with a maleimide conjugation functional group site (Ab-(G4S15)(4), Ab-(G5S29)(4)) are also described. The in vitro molar relaxivity of the Ab-(G4S15)(4) conjugate, measured at pH 7.4, 22 °C, and 3T showed a moderate increase in relaxivity as compared to Magnevist (6.7 vs 4.0 mM(-1) s(-1)) while the Ab-(G5S29)(4) conjugate was 2-fold higher (9.1 vs 4.0 mM(-1) s(-1)). The data showed that only a high injection dose (0.050 mmol Gd(3+)/kg) produced a detectable contrast enhanced contrast for the Ab-(G4S15)(4) conjugate while a lower dose (0.035 mmol Gd(3+)/kg) was sufficient for the Ab-(G5S29)(4) conjugate. The antibody-SMCC conjugate was purified by a Sephadex G-100 column, and the antibody-dendrimer-based agents were purified by spin filtration using a Centricon filter (50,000 MCO). The protein assay coupled with cysteine and Ellman's assay indicated an antibody to dendrimer ratio of 1:4. The in vivo blood clearance half-lives of the four agents measured at the jugular vein were ~12-22 min.

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Figures

Figure 1
Figure 1
Conjugation of 1 to a dendrimer.
Figure 2
Figure 2
Conjugation of a mono-thiol half-dendrimer to F(ab’)2 of panitumumab.
Figure 3
Figure 3
Molar relaxivity plot of Magnevist™ (◆; 4.1 mM-1s-1), Ab-(G4S15)4 (▲; 6.7 mM-1s-1), Ab-(G5S29)4 (■; 9.1 mM-1s-1), G4SS30 (+; 16.5 mM-1s-1), G5SS58 (–; 27.2 mM-1s-1) measured at room temperature and 3T.
Figure 4
Figure 4
1/T1 NMRD profiles of 1 mM solution of Magnevist® (◆), Ab-(G4S15)4 (×), Ab-(G5S29)4 (–), G4SS30 (■),G5SS58 (▲) as a function of frequencies at 23°C.
Figure 5
Figure 5
Dynamic MRI of mice injected with 0.015 mmol/kg of G4SS30 and G5SS58, and 0.050 mmol/kg of Ab-(G4S15)4, and 0.035 mmol/kg of Ab-(G5S29)4. All images were acquired at 12-16 min post injection. A pre-contrast image was included for comparison.
Figure 6
Figure 6
Average blood clearance rates measured at the jugular vein of G4SS30, G5SS58, Ab-(G4S15)4 and Ab-(G5S29)4.
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