Biotin Reagents for Antibody Pretargeting. 3. Synthesis, Radioiodination, and Evaluation of Biotinylated Starburst Dendrimers

Abstract

We are investigating the hypothesis that biotin multimers can be used with streptavidin and monoclonal antibody conjugates in cancer pretargeting protocols to provide a method of increasing the amount of radioactivity bound on cancer cells in patients. As part of that investigation, a series of biotinylated Starburst dendrimers (BSBDs) have been prepared and evaluated in vitro and in vivo. In this study, a new biotinidase-stabilized, water-solubilizing biotinylation reagent was prepared and reacted with Starburst (PAMAM) dendrimers, generations 0, 1, 2, 3, and 4. The reaction conditions employed resulted in perbiotinylation of generation 0 (four biotin moieties conjugated), generation 1 (eight biotin moieties conjugated), generation 2 (16 biotin moieties conjugated), and generation 3 (32 biotin moieties conjugated). With generation 4, incomplete biotinylation was achieved resulting in the largest portion of that BSBD having 51 biotin moieties (of 64 possible) conjugated. The ability of each BSBD to cross-link streptavidin (SAv) was examined in an in vitro assay. In that assay, an assessment was made of the quantity of [125I]SAv bound with polystyrene-bound SAv after treatment with the synthesized BSBDs. All BSBDs cross-linked the polystyrene-bound SAv with [125I]SAv; however, the amount of [125I]SAv bound varied with the different BSBDs. Roughly 1 equiv of [125I]SAv was bound when Starburst dendrimers containing three or four biotin moieties (generation 0) were used. Two equivalents were bound with BSBD generation 1, and 4 equiv were bound with BSBDs generations 2, 3, and 4. To assess the distribution of BSBDs generations 0, 1, and 2 in mice (at 4 h postinjection), a method was developed for radioiodinating them using the NHS ester of p-[125I]iodobenzoate ([125I]PIB). It was found that the radioiodinated BSBDs had low blood concentrations (i.e., 0.13-0.20% ID/g) at the 4 h time point. In fact, most tissues examined had low concentrations of biotinylated dendrimers, except kidney and liver. Kidney had the highest concentration of [125I]-labeled BSBDs, and its concentration increased with increasing size and charge of dendrimer (e.g., 8-48% ID/g). On the basis of the increased radioactivity observed in the in vitro assay and the rapid clearance from blood in mice, additional in vivo studies with perbiotinylated Starburst dendrimer, generation 2, are planned.

Nanoparticle Targeting of Anticancer Drug Improves Therapeutic Response in Animal Model of Human Epithelial Cancer

“…Radiolabeled compounds were synthesized from G5-(Ac) 50 -(FA) 6 or G5-(Ac) 50 using tritiated acetic anhydride (Ac-3 H) as described previously (18,21,23). The tritiated conjugates, G5-3 H-FA and G5-3 H, were fully acetylated.…”

Influence of Surface Functionality of Poly(propylene imine) Dendrimers on Protease Resistance and Propagation of the Scrapie Prion Protein

“…Amino-terminated dendrimers are reported to have limited potential for application in therapy due to their generation and dose-dependent toxicity profile [53][54][55] . The functionalisation of their peripheral groups has been reported to improve their toxic properties 56 .…”

“…Glycosylation of these dendrimers was found to further increase the permeation 65 . Interestingly, toxicity of uncharged and/or glycosylated dendrimers is lower than cationic dendrimers 38,[53][54][55] . In agreement with these reports, there was little cytotoxicity observed for PPI glycodendrimers in this study.…”

Influence of dendrimers on red blood cells

“…However, as their interactions with cell components are non-selective, dendrimers also have a potential to cause cyto-and haemotoxicity due to their terminal (e.g. amino) groups and multiple cationic charge [12,19]. Folic acid is often conjugated to dendrimers to increase the target specificity of these nanoparticles and facilitate dendrimers' location near the tumour, since in various cancer cell lines folic acid receptor are overexpressed [20,21].…”