Revisiting the value of competition assays in folate receptor-mediated drug delivery

Abstract

Polymeric nanoparticles have been studied for gene and drug delivery. These nanoparticles can be modified to utilize a targeted delivery approach to selectively deliver their payload to specific cells, while avoiding unwanted delivery to healthy cells. One commonly over-expressed receptor which can be targeted by ligand-conjugated nanoparticles is the folate receptor alpha (FRα). The ability to target FRα remains a promising concept, and therefore, understanding the binding dynamics of the receptor with the ligand of the nanoparticle therapeutic can provide valuable insight. This manuscript focuses on the interaction between self-assembled nanoparticles decorated with a folic acid (FA) ligand and FRα. The nanoparticles consist of micelles formed with a FA conjugated triblock copolymer (PEI-g-PCL-b-PEG-FA) which condensed siRNA to form micelleplexes. By combining biological and biophysical approaches, this manuscript explores the binding kinetics and force of the targeted siRNA containing nanoparticles to FRα in comparison with free FA. We demonstrate via flow cytometry and atomic force microscopy that multivalent micelleplexes bind to FRα with a higher binding probability and binding force than monovalent FA. Furthermore, we revisited why competitive inhibition studies of binding of multivalent nanoparticles to their respective receptor are often reported in literature to be inconclusive evidence of effective receptor targeting. In conclusion, the results presented in this paper suggest that multivalent targeted nanoparticles display strong receptor binding that a monovalent ligand may not be able to compete with under in vitro conditions and that high concentrations of competing monovalent ligands can lead to measurement artifacts.

Coming in and Finding Out: Blending Receptor‐Targeted Delivery and Efficient Endosomal Escape in a Novel Bio‐Responsive siRNA Delivery System for Gene Knockdown in Pulmonary T Cells

“…[36] This concentration-dependent influence of additional free Tf molecules competing for the binding sites at the TfR supports the anticipated hypothesis that both Tf-PEI and Tf-Mel-PEI polyplexes are taken up via TfR mediated endocytosis. While uptake of those polyplexes without the targeting ligand, as expected, was not influenced by the presence of free transferrin, the Tf-targeted polyplexes exhibited clearly compromised uptake efficiencies, although the uptake was not blocked completely.…”

“…[36] The lowered association rate observed here for polyplexes may be caused by steric hindrance of PEI resulting in a reduced accessibility of Tf in the particles compared to the free ligand. Comparison of binding affinities revealed that Tf-PEI particles bind even more stable than free transferrin, showing an overall comparable dissociation rate.…”

Novel functionalized nanoparticles for tumor-targeting co-delivery of doxorubicin and siRNA to enhance cancer therapy

“…23 Briefly, 1 mg of the RGDfC-SeNPs@DOX/siRNA nanoparticles was suspended in 1 mL of phosphate-buffered saline (PBS) in DEPC and transferred into a dialysis tube. Afterwards, the tubes were immersed in 49 mL of buffer solution at pH 5.4 or pH 7.4 under horizontal shaking (100 rpm).…”

In vitro and in vivo delivery of siRNA via VIPER polymer system to lung cells

“…In order to measure polyplex accumulation following pulmonary delivery, a previously described mouse model was used with slight modifications. [16] Female balb/c mice were purchased from Charles River Laboratories (Boston, MA, US) and used at 5 weeks’ age. Briefly, mice were intratracheally instilled (under ketamine/xylazine anesthesia) with 50 µl of CP, VIPER or PEI polyplexes, prepared with 2 nmol TYE™-563 (IDT) or pHrodo™ (Thermo Fisher Scientific) labeled siRNA at N/P 8.…”