Surface conjugation of antibodies improves nanoparticle uptake in bronchial epithelial cells

Abstract

Background: Advances in Molecular Therapy have made gene editing through systemic or topical administration of reagents a feasible strategy to treat genetic diseases in a rational manner. Encapsulation of therapeutic agents in nanoparticles can improve intracellular delivery of therapeutic agents, provided that the nanoparticles are efficiently taken up within the target cells. In prior work we had established proof-of-principle that nanoparticles carrying gene editing reagents can mediate site-specific gene editing in fetal and adult animals in vivo that results in functional disease improvement in rodent models of β-thalassemia and cystic fibrosis. Modification of the surface of nanoparticles to include targeting molecules (e.g. antibodies) holds the promise of improving cellular uptake and specific cellular binding.

Methods and findings: To improve particle uptake for diseases of the airway, like cystic fibrosis, our group tested the impact of nanoparticle surface modification with cell surface marker antibodies on uptake in human bronchial epithelial cells in vitro. Binding kinetics of antibodies (Podoplanin, Muc 1, Surfactant Protein C, and Intracellular Adhesion Molecule-1 (ICAM)) were determined to select appropriate antibodies for cellular targeting. The best target-specific antibody among those screened was ICAM antibody. Surface conjugation of nanoparticles with antibodies against ICAM improved cellular uptake in bronchial epithelial cells up to 24-fold.

Conclusions: This is a first demonstration of improved nanoparticle uptake in epithelial cells using conjugation of target specific antibodies. Improved binding, uptake or specificity of particles delivered systemically or to the luminal surface of the airway would potentially improve efficacy, reduce the necessary dose and thus safety of administered therapeutic agents. Incremental improvement in the efficacy and safety of particle-based therapeutic strategies may allow genetic diseases such as cystic fibrosis to be cured on a fundamental genetic level before birth or shortly after birth.

Fig 1

CFBE Cells that were A) Unstained B&C) Mouse and Rabbit Isotype controls and stained for: D) Surfactant Protein C, E) Podoplanin, F&G) Two different Muc 1 (CD227) antibody clones, and H) Intracellular Adhesion Molecule 1 (ICAM). 40X Confocal image. (Blue = DAPI nuclear stain) (Bar = 20 micron).

Fig 2. Antibody Binding Curves of antibody concentration (x axis) versus Mean Fluorescence Intensity (MFI) (y axis) demonstrating receptor density (B_max) and antibody-antigen affinity (K_d).

A) Surfactant Protein C (SFTPC) B) Podoplanin (PDPN) (SFTPC and PDPN did not fit a curve, thus B_max and Kd calculations were not possible). C) Muc 1 D) CD 227 (second clone of Muc 1 antibody) E) Intracellular Adhesion Molecule 1 (ICAM). F) Comparison of MFI distribution at an antibody concentration of 30 nM of each antibody tested.

Fig 3. Nanoparticles synthesis with surface conjugation to antibody improves uptake in CFBE cells in vitro.

A) By SEM, PLA-PEG particles are uniform in size (Bar = 1μm). B&C) Flow cytometry of CFBE Cells treated with green (DiO) dye. These NPs were either, blank unconjugated, isotype conjugated or ICAM antibody conjugated. B MFI of treated cells and C) percentage cells with NP internalization were measured. (n = 3 for each treatment) (*** p<0.001 & **** p<0.0001).