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. 2015 Apr 1:10:2595-617.
doi: 10.2147/IJN.S79367. eCollection 2015.

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

Affiliations

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

Christian Ndong et al. Int J Nanomedicine. .

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.

Keywords: antibody fragment; biodistribution; nanoparticle targeting; ovarian cancer.

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Figures

Figure 1
Figure 1
Schematic of workflow for fab and IONP functionalization. Notes: Monomeric Ffab and Bfab are subjected to reduction/activation using 20 mM cysteine followed by conjugation with maleimiede-PEG2-biotin or maleimide-activated IONPs. Two IONP types were examined in this study: Dartmouth CMD and commercial BNF, which were functionalized with maleimide groups using EDC and N-(2-aminoethyl) maleimide, or sulfo-GMBS, respectively. Abbreviations: IONP, iron oxide nanoparticle; fab, an engineered monoclonal antibody fragment; Ffab, Farletuzufab, engineered from monoclonal antibody Farletuzumab; Bfab, anti-botulinum toxin fab fragment; PEG2, polyethylene glycol 2; CMD, carboxymethyl-dextran; BNF, bionized nanoferrite; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carboiimide; sulfo-GMBS, N-(γ-maleimobutyryloxy) sulfosuccinimide ester.
Figure 2
Figure 2
Characterization of Ffab and Bfab antibody fragments. Notes: Reducing SDS-PAGE gels of (A) purified Ffab and (B) purified Bfab, stained with Coomassie brilliant blue. Non-reducing SDS-PAGE gel of (C) purified Ffab and (D) purified Bfab, stained with Coomassie brilliant blue. Lane 1 represents size exclusion-purified Ffab or Bfab, lane 2 is Ffab or Bfab after cysteine reductive activation, and lane 3 is Ffab or Bfab after maleimide-PEG2-biotin conjugation. Abbreviations: SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; fab, an engineered monoclonal antibody fragment; Ffab, Farletuzufab, engineered from monoclonal antibody Farletuzumab; Bfab, Botulifab anti-botulinum toxin fab fragment; PEG2, polyethylene glycol 2.
Figure 3
Figure 3
Binding analysis of Ffab and Bfab antibody fragments. Notes: (A) Representative ELISA binding profile of Ffab (closed square) and Bfab (open circle) with rFOLRα protein. (B) Representative cell-based ELISA for Ffab (closed symbols) and Bfab (open symbols) binding to FOLR+/HER2+ KB cells (squares) or FOLR/HER2+ SKBR3 cells (triangles). Error bars represent standard deviation from technical triplicates. Abbreviations: fab, an engineered monoclonal antibody fragment; Ffab, Farletuzufab, engineered from monoclonal antibody Farletuzumab; Bfab, Botulifab anti-botulinum toxin fab fragment; ELISA, enzyme-linked immunosorbent assay; FOLR, folate receptor; rFOLRα, recombinant folate receptor alpha; HER2, human epidermal growth factor receptor 2.
Figure 4
Figure 4
In vitro binding studies of Ffab-IONPs and Bfab-IONPs. Notes: (A) Dose–response binding curves for Ffab-CMD (closed square) and Bfab-CMD (open circles) with rFOLRα protein. (B) Dose–response binding curves for Ffab-BNF (closed square) and Bfab-BNF (open circles) with rFOLRα protein. (C) Binding of FOLRα+ KB cancer cells by Ffab-CMD and Bfab-CMD dosed at 35 μg/mL (0.6 nM). (D) Binding of FOLRα+ KB cancer cells by Ffab-BNF and Bfab-BNF dosed at 35 μg/mL (0.035 nM). Error bars represent standard deviation from technical triplicates. ***P<0.001, two-tailed unpaired t-test. Abbreviations: fab, an engineered monoclonal antibody fragment; Ffab, Farletuzufab, engineered from monoclonal antibody Farletuzumab; Bfab, Botulifab anti-botulinum toxin fab fragment; IONP, iron oxide nanoparticle; CMD, carboxymethyl-dextran; rFOLRα, recombinant folate receptor alpha; BNF, bionized nanoferrite, NP, nanoparticle.
Figure 5
Figure 5
TEM imaging of Ffab-IONP and Bfab-IONP subcellular localization following in vitro binding to FOLRα+ KB cancer cells. Notes: Magnification of 25,000× showing the binding of (A) Ffab-CMD and (D) Ffab-BNF on the KB cell surface (arrows). Magnification of 10,000× showing (B) Ffab-CMD and (E) Ffab-BNF within intracellular vesicles (arrows). Magnification of 25,000× showing no evidence of nanoparticles on KB tumor cells treated with (C) Bfab-CMD or (F) Bfab-BNF. Scale bars are 100 nm (A, C, D and F) and 500 nm (B and E). Abbreviations: TEM, transmission electron microscopy; fab, an engineered monoclonal antibody fragment; Ffab, Farletuzufab, engineered from monoclonal antibody Farletuzumab; Bfab, Botulifab anti-botulinum toxin fab fragment; IONP, iron oxide nanoparticle; CMD, carboxymethyl-dextran; rFOLRα, recombinant folate receptor alpha; BNF, bionized nanoferrite; NP, nanoparticle.
Figure 6
Figure 6
In vivo biodistribution of IONPs following IP administration. Notes: Total iron content of various tissue compartments is shown: (A) tumor; (B) fat; (C) liver; (D) spleen; and (E) kidney. Data obtained by ICP-MS from five mice per group approximately 18 hours post-injection. Statistical significance was analyzed by one-way ANOVA with a Tukey multiple comparison post-test, and the results of individual comparisons are provided in the tables below each graph. ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05. Abbreviations: PBS, phosphate-buffered saline; fab, an engineered monoclonal antibody fragment; Ffab, Farletuzufab, engineered from monoclonal antibody Farletuzumab; Bfab, Botulifab anti-botulinum toxin fab fragment; CMD, carboxymethyl-dextran; rFOLRα, recombinant folate receptor alpha; BNF, bionized nanoferrite; IONPs, iron oxide nanoparticles; IP, intraperitoneal; ICP-MS, inductively coupled plasma mass spectrometry; ANOVA, analysis of variance.
Figure 6
Figure 6
In vivo biodistribution of IONPs following IP administration. Notes: Total iron content of various tissue compartments is shown: (A) tumor; (B) fat; (C) liver; (D) spleen; and (E) kidney. Data obtained by ICP-MS from five mice per group approximately 18 hours post-injection. Statistical significance was analyzed by one-way ANOVA with a Tukey multiple comparison post-test, and the results of individual comparisons are provided in the tables below each graph. ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05. Abbreviations: PBS, phosphate-buffered saline; fab, an engineered monoclonal antibody fragment; Ffab, Farletuzufab, engineered from monoclonal antibody Farletuzumab; Bfab, Botulifab anti-botulinum toxin fab fragment; CMD, carboxymethyl-dextran; rFOLRα, recombinant folate receptor alpha; BNF, bionized nanoferrite; IONPs, iron oxide nanoparticles; IP, intraperitoneal; ICP-MS, inductively coupled plasma mass spectrometry; ANOVA, analysis of variance.
Figure 7
Figure 7
Histology of excised tumors from ovarian cancer model. Notes: Representative images where the tumor and peritoneal membrane are indicated. HE of tumor sections from animals treated with (A) PBS, (B) Bfab-BNF, or (C) Ffab-BNF. (D, E, F) Prussian blue staining of the same slides, respectively, to identify IONPs. Abbreviations: HE, hematoxylin eosin; PBS, phosphate-buffered saline; fab, an engineered monoclonal antibody fragment; Ffab, Farletuzufab, engineered from monoclonal antibody Farletuzumab; Bfab, Botulifab anti-botulinum toxin fab fragment; IONPs, iron oxide nanoparticles; BNF, bionized nanoferrite.

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