Antibody-mediated targeting of iron oxide nanoparticles to the folate receptor alpha increases tumor cell association in vitro and in vivo

Abstract

Active molecular targeting has become an important aspect of nanoparticle development for oncology indications. Here, we describe molecular targeting of iron oxide nanoparticles (IONPs) to the folate receptor alpha (FOLRα) using an engineered antibody fragment (Ffab). Compared to control nanoparticles targeting the non-relevant botulinum toxin, the Ffab-IONP constructs selectively accumulated on FOLRα-overexpressing cancer cells in vitro, where they exhibited the capacity to internalize into intracellular vesicles. Similarly, Ffab-IONPs homed to FOLRα-positive tumors upon intraperitoneal administration in an orthotopic murine xenograft model of ovarian cancer, whereas negative control particles showed no detectable tumor accumulation. Interestingly, Ffab-IONPs built with custom 120 nm nanoparticles exhibited lower in vitro targeting efficiency when compared to those built with commercially sourced 180 nm nanoparticles. In vivo, however, the two Ffab-IONP platforms achieved equivalent tumor homing, although the smaller 120 nm IONPs were more prone to liver sequestration. Overall, the results show that Ffab-mediated targeting of IONPs yields specific, high-level accumulation within cancer cells, and this fact suggests that Ffab-IONPs could have future utility in ovarian cancer diagnostics and therapy.

A Feasibility Study of Nonlinear Spectroscopic Measurement of Magnetic Nanoparticles Targeted to Cancer Cells

“…In this study 110 – 120 nm (20-40 nm iron core) MNPs were functionalized with a Fab fragment of the Farletuzumab antibody (Ffab), which specifically binds to the alpha folate receptor cell surface marker (αFR). These Ffab-MNP conjugates have previously been shown to selectively bind to cancer cells that overexpress αFR, whereas negative control MNP targeted to botulinum toxin fail to exhibit any cellular association under the same conditions [42]. In Fig 2, we show the measured quantities of bound and unbound MNPs in the KB and Igrov human cancer cell lines.…”

“…Farletuzumab Fab antibody fragment (Ffab) was purified and characterized as described earlier [42]. Cysteine-reduced Ffab was added to the maleimide-functionalized CMD at a 1:10 (w/w) ratio and incubated at room temperature for 16 hours at 4 °C on a shaker set at 125 rpm.…”

“…All processes were performed in a sterile environment using sterile and endotoxin free buffers. Micro BCA assay (Thermo Scientific, Rockford, IL) was used to determine the amount of Ffab covalently bound to MNP, as described previously [42]. …”

“…Cells were incubated at 37 °C, 5% CO 2 with gentle shaking (125 rpm) for 4 hours. Antibody targeted CMD MNPs have been shown previously to be stable for more than 8 hours under these conditions [42]. Cells were centrifuged at 1200 rpm for 10 minutes and the supernatant containing the unbound Ffab-CMD nanoparticles was collected for a ferrozine assay as described earlier [42].…”

“…Antibody targeted CMD MNPs have been shown previously to be stable for more than 8 hours under these conditions [42]. Cells were centrifuged at 1200 rpm for 10 minutes and the supernatant containing the unbound Ffab-CMD nanoparticles was collected for a ferrozine assay as described earlier [42]. Briefly, 300 μl of HCl 1.4 M was added to 20 μl supernatant in a 48-well plate (Corning, Tewksbury, MA) and incubated at 70 °C for 2 hours.…”