PAMAM dendrimer-based contrast agents for MR imaging of Her-2/neu receptors by a three-step pretargeting approach

A three-step pretargeting approach pioneered by Paganelli et al. (1) about 20 years ago using biotin-avidin systems still holds promise in increasing the tumor/nontumor ratio compared to a direct targeting approach (2). While more popular in the delivery of radionuclides for imaging and therapy, the low concentration of receptors and the intrinsic low sensitivity of MRI have limited applications of pretargeting in MRI. Her-2/neu overexpressing breast tumors are a good model system for an MRI pretargeting approach due to the large number of receptors expressed uniformly on the cancer cell surface and the availability of trastuzumab, an FDA-approved antibody against the Her-2/neu receptors (3,4). Success in delivering MRI agents by pretargeting can also be extended to therapy, which is important as the amplification and overexpression of Her-2/neu, a member of the epidermal growth factor receptor (EGFR) family of tyrosine kinases that regulates cell proliferation and differentiation, has been linked to poor prognosis in several human cancers such as breast, prostate, lung, ovary, and colon cancers. Because of its low intrinsic sensitivity, the success of MRI depends on the development of highly efficient contrast agents. For example, it was demonstrated that conjugating of Gd(III) chelates to high molecular weight carriers is highly beneficial for in vivo MR imaging as it provides 1) prolonged intravascular circulation time that results in improved pharmacokinetics of the agent at the target site, and 2) slows down molecular rotation that translates to increased T1 relaxivity at high magnetic field typically used in preclinical MRI studies (5). Macromolecular MRI contrast agents based on PAMAM dendrimers conjugated with a diethylenetriamimepentaacetic acid (DTPA) derivative, 2–(p-isothiocyanatobenzyl)– 6–methyl–diethylenetriaminepentaacetic acid (1B4M), to chelate gadolinium metal ions have been proven to be versatile in MRI applications due to their mono-disperse molecular size and the ability to fine-tune molecular weights by using dendrimers of different generations (6–8). In our studies we chose a PAMAM generation 4 dendrimer (G4D) due to its i) 64 surface functional amine groups (−NH2) that can be easily modified, ii) moderate blood circulation time, and iii) relatively fast excretion via the kidney (7). It was reported that a G4-based gadolinium agent was quickly excreted via the kidney primarily during the first pass, while exhibiting no measurable leakage from normal blood vessels (6). The surface amine groups of PAMAM dendrimers are positively charged at physiological pH. Microvascular endothelium is lined with a glycocalyx layer which is composed of negatively charged sulfated glycosaminoglycans (9,10). Electric static interaction between the positive amine group and the negative endothelium promotes faster clearance as well as nonspecific binding of the PAMAM dendrimer. Cationic macromolecules also show higher glomerular permeation than anionic macromolecules of similar molecular weights, as glomerular capillary walls function as a charge selective barrier having negative charge (11). Conjugation of each G4D −NH2 group to diethylenetriaminepentaacetic acid (DTPA) chelating gadolinium(III) (DTPA-Gd) resulted in a net charge of −1, helping to reduce the overall positive charge of the final complex G4D-(DTPA-Gd)n. Local positive charges due to the remaining free surface amine groups that were not conjugated to (DTPA-GD) can be further reduced by succinylation with succinic anhydride. In the present study, athymic mice bearing Her-2/neu overexpressing human breast tumor BT-474 xenografts were imaged through a three-step pretargeting approach that included biotinylated trastuzumab, avidin, and bG4D-Gd. To test the effect of the surface charge of bG4D-Gd on blood circulation time and kidney excretion we also used its succinylated form, bG4D-Gd-SA.