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

Ndong C, Toraya-Brown S, Kekalo K, Baker I, Gerngross TU, Fiering SN, Griswold KE - Int J Nanomedicine (2015)

Bottom Line: Here, we describe molecular targeting of iron oxide nanoparticles (IONPs) to the folate receptor alpha (FOLRα) using an engineered antibody fragment (Ffab).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.

View Article: PubMed Central - PubMed

Affiliation: Thayer School of Engineering, Dartmouth, Hanover, NH, USA.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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f2-ijn-10-2595: 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.

Mentions: Ffab and Bfab purities were evaluated by SDS-PAGE. Under reducing conditions (presence of 50 mM dithiothreitol), the heavy and light chains of both Ffab and Bfab migrated as a single 25 kDa band (Figure 2A, B; lane 1). In non-reducing conditions (no dithiothreitol), Ffab and Bfab migrated predominantly as the expected 48 kDa band, although less intense low molecular weight bands corresponding to free heavy chain and light chain were also observed (Figure 2C, D; lane 1). Following reductive activation of both Ffab and Bfab, much of the intact fab band at 48 kDa was shifted to the lower molecular weight bands for free heavy chain and light chain (Figure 2C, D; lane 2), which was consistent with the appearance of these species during LC-MS analysis. Upon conjugation to maleimide-PEG2-biotin, substantial portions of the intact fabs were reconstituted, although bands for free heavy and light chain remained evident (Figure 2C, D; lane 3). The LC-MS analysis had identified heavy and light chain species in which the cysteines involved in interchain disulfide bond formation had been capped with maleimide-PEG2-biotin, and the corresponding free heavy and free light chain bands in the SDS-PAGE of biotinylated fabs corroborated that result (ie, maleimide-capped light and heavy chains are unable to reform intrachain disulfide bonds). We emphasize again that previous studies have shown that analogous, reduced yet intact fabs retain the binding activity and efficiency of their disulfide bonded counterparts.26,29


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

Ndong C, Toraya-Brown S, Kekalo K, Baker I, Gerngross TU, Fiering SN, Griswold KE - Int J Nanomedicine (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4388088&req=5

f2-ijn-10-2595: 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.
Mentions: Ffab and Bfab purities were evaluated by SDS-PAGE. Under reducing conditions (presence of 50 mM dithiothreitol), the heavy and light chains of both Ffab and Bfab migrated as a single 25 kDa band (Figure 2A, B; lane 1). In non-reducing conditions (no dithiothreitol), Ffab and Bfab migrated predominantly as the expected 48 kDa band, although less intense low molecular weight bands corresponding to free heavy chain and light chain were also observed (Figure 2C, D; lane 1). Following reductive activation of both Ffab and Bfab, much of the intact fab band at 48 kDa was shifted to the lower molecular weight bands for free heavy chain and light chain (Figure 2C, D; lane 2), which was consistent with the appearance of these species during LC-MS analysis. Upon conjugation to maleimide-PEG2-biotin, substantial portions of the intact fabs were reconstituted, although bands for free heavy and light chain remained evident (Figure 2C, D; lane 3). The LC-MS analysis had identified heavy and light chain species in which the cysteines involved in interchain disulfide bond formation had been capped with maleimide-PEG2-biotin, and the corresponding free heavy and free light chain bands in the SDS-PAGE of biotinylated fabs corroborated that result (ie, maleimide-capped light and heavy chains are unable to reform intrachain disulfide bonds). We emphasize again that previous studies have shown that analogous, reduced yet intact fabs retain the binding activity and efficiency of their disulfide bonded counterparts.26,29

Bottom Line: Here, we describe molecular targeting of iron oxide nanoparticles (IONPs) to the folate receptor alpha (FOLRα) using an engineered antibody fragment (Ffab).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.

View Article: PubMed Central - PubMed

Affiliation: Thayer School of Engineering, Dartmouth, Hanover, NH, USA.

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.

No MeSH data available.


Related in: MedlinePlus