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

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.

Figure 1

Conjugation of 1 to a dendrimer.

Figure 2

Conjugation of a mono-thiol half-dendrimer to F(ab’)₂ of panitumumab.

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

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

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

Average blood clearance rates measured at the jugular vein of G4SS30, G5SS58, Ab-(G4S15)₄ and Ab-(G5S29)₄.