Vascular Targeting of Radiolabeled Liposomes with Bio-Orthogonally Conjugated Ligands: Single Chain Fragments Provide Higher Specificity than Antibodies

Abstract

Liposomes are a proven, versatile, and clinically viable technology platform for vascular delivery of drugs and imaging probes. Although targeted liposomes have the potential to advance these applications, complex formulations and the need for optimal affinity ligands and conjugation strategies challenge their translation. Herein, we employed copper-free click chemistry functionalized liposomes to target platelet-endothelial cell adhesion molecule (PECAM-1) and intracellular adhesion molecule (ICAM-1) by conjugating clickable monoclonal antibodies (Ab) or their single chain variable fragments (scFv). For direct, quantitative tracing, liposomes were surface chelated with 111In to a >90% radiochemical yield and purity. Particle size and distribution, stability, ligand surface density, and specific binding to target cells were characterized in vivo. Biodistribution of liposomes after IV injection was characterized in mice using isotope detection in organs and by noninvasive imaging (single-photon emission computed tomography/computed tomography, SPECT/CT). As much as 20–25% of injected dose of liposomes carrying PECAM and ICAM ligands, but not control IgG accumulated in the pulmonary vasculature. The immunospecificity of pulmonary targeting of scFv/liposomes to PECAM-1 and ICAM-1, respectively, was 10-fold and 2.5-fold higher than of Ab/liposomes. Therefore, the combination of optimal ligands, benign conjugation, and labeling yields liposomal formulations that may be used for highly effective and specific vascular targeting.

Supramolecular Organization Predicts Protein Nanoparticle Delivery to Neutrophils for Acute Lung Inflammation Diagnosis and Treatment

“…The variety of protein nanostructures accumulating in inflamed lungs in our data includes particles that have been investigated as targeted drug delivery vehicles where marginated neutrophils are not the intended site of accumulation. 36,38,47,48,62 The patterns in our data indicate that future studies may reveal additional nanoparticles that accumulate in the lungs following inflammatory insult. This study therefore serves as evidence that inflammatory challenges may prompt profound off-target changes in the biodistributions of nanomaterials, including dramatic shunting of nanoparticles and any associated drug payload to the lungs.…”

“…Liposomes were functionalized with rat IgG conjugated via SATA-maleimide chemistry (SATA-IgG liposomes) or via recently demonstrated copper-free click chemistry methods. 47 Briefly, click chemistry methods entailed NHS-ester conjugation of an excess of strained alkyne (dibenzocyclooctyne, DBCO) to IgG, followed by reaction of the DBCO-functionalized IgG with liposomes containing PEG-azide-terminated lipids (DBCO-IgG liposomes, Figure 4A). DBCO-IgG liposomes had a diameter of 128.3±4.3 nm and a PDI of 0.17±0.03 and SATA-IgG liposomes had a diameter of 178.8±6.9 nm and a PDI of 0.23±0.03 (Supplementary Figure 1C).…”

“…Azide-functionalized liposomes were prepared by thin film hydration techniques, as previously described. 47 The lipid film was composed of 58 mol% DPPC (1,2dipalmitoyl-sn-glycero-3-phosphocholine), 40 mol% cholesterol, and 2 mol% azide-PEG2000-DSPE (all lipids from Avanti). 0.5 mol% Top Fluor PC (1-palmitoyl-2-(dipyrrometheneboron difluoride) undecanoyl-sn-glycero-3-phosphocholine) was added to prepare fluorescent liposomes.…”

“…Nanoparticle Labeling with 111 In 111 In labeling of nanoparticles followed previously described methods, with adaptation for new particles. 47 All radiolabeling chelation reactions were performed using metal free conditions to prevent contaminating metals from interfering with chelation of 111 In by DTPA or DOTA. Metals were removed from buffers using Chelex 100 metal affinity resin (Biorad, Laboratories, Hercules CA).…”

“…As described previously, 47 thirty minutes after injection of 80 µCi of 111 In-labeled nanogels, anesthetized mice were sacrificed by cervical dislocation. Mice were placed into a MiLabs U-SPECT (Utrecht, Netherlands) scanner bed.…”