Uptake Of Fluorescent Nanoparticles Research Articles

Facile Rebridging Conjugation Approach to Attain Monoclonal Antibody-Targeted Nanoparticles with Enhanced Antigen Binding and Payload Delivery.

Adopting conventional conjugation approaches to construct antibody-targeted nanoparticles (NPs) has demonstrated suboptimal control over the binding orientation and the structural stability of monoclonal antibodies (mAbs). Hitherto, the developed antibody-targeted NPs have shown proof of concept but lack product homogeneity, batch-to-batch reproducibility, and stability, precluding their advancement toward the clinic. To circumvent these limitations and advance toward clinical application, herein, a refined approach based on site-specific construction of mAb-immobilized NPs will be appraised. Initially, the conjugation of atezolizumab (anti-PDL1 antibody, Amab) with polymeric NPs was developed using bis-haloacetamide (BisHalide) rebridging chemistry, followed by click chemistry (NP-Fab BisHalide Ab and NP-Fc BisHalide Ab). For comparison purposes, mAb-immobilized NPs developed utilizing conventional conjugation methods, namely, N-hydroxysuccinimide (NHS) coupling and maleimide chemistry (NP-NHS Ab and NP-Mal Ab), were included. Next, flow cytometry and confocal microscopy experiments evaluated the actively targeted NPs (loaded with fluorescent dye) for cellular binding and uptake. Our results demonstrated the superior and selective binding and uptake of NP-Fab BisHalide Ab and NP-Fc BisHalide Ab into EMT6 cells by 19-fold and 13-fold, respectively. To evaluate the PDL1-dependent cell uptake and the selectivity of the treatments, a blocking step of the PDL1 receptor with Amab was performed prior to incubation with NP-Fab BisHalide Ab and NP-Fc BisHalide Ab. To our delight, the binding and uptake of fluorescent NPs were reduced significantly by 3-fold for NP-Fab BisHalide Ab, demonstrating the PDL1-mediated uptake. Moreover, NP-Fab BisHalide Ab and NP-Fc BisHalide Ab were entrapped with the paclitaxel payload, and their cytotoxicity was evaluated. They showed significant enhancements compared to free paclitaxel and NP-NHS Ab. Overall, this work will provide a facile conjugation method that could be implemented to actively target NPs with a plethora of therapeutic mAbs approved for various malignancies.

Production, Physico-Chemical Characterization and Biodistribution Studies of Lipid Nanoparticles

This study describes the preparation, characterization, in vitro uptake and in vivo biodistribution in mice of solid lipid nanoparticles and nanostructured lipid carriers. The effect of nanoparticle lipid matrix, presence of fluorescent and functionalization by polysorbate 80 on dimensional distribution and morphology have been studied by sedimentation field flow fractionation, photon correlation spectroscopy and cryogenic transmission electron microscopy. The complementary use of different techniques demonstrated that lipid matrix composition, presence of fluorescent dye and polysorbate 80 functionalization have little effect on nanoparticle morphology and size distribution. Uptake of fluorescent nanoparticles was determined in vitro by human brain endothelial cells, showing that nanoparticles treated by polysorbate 80 displayed lower uptake values with respect to the corresponding control nanoparticles. Biodistribution of solid lipid nanoparticles treated by polysorbate 80 was evaluated by fluorescent luminescent imaging after intraperitoneal administration in mice. The in vivo images indicate that nanoparticles were able to reach the brain, even if they prevalently accumulated in liver and spleen.

Abstract 5463: Dual Channel Hybrid Fluorescence Molecular Tomography/X-ray Computed Tomography (FMT-CT) of Inflammation in Murine Atherosclerotic Plaques

Monocyte/macrophages play a key role in the development and progression of atherosclerotic lesions. Here we present a novel hybrid in vivo imaging approach that simultaneously reports on lesion anatomy, phagocytic activity by uptake of fluorescent nanoparticles and extracellular matrix remodeling with a protease-activatable agent, detectable with hybrid FMT-CT imaging. We co-injected 5 aged apoE deficient and 5 wild type mice with 15mg/kg of macrophage-avid nanoparticle (CLIO-680, emission 700nm) and 2nmol of a cathepsin-activatable protease reporter (Prosense-750, emission 780nm). Mice were imaged using a free space dual channel FMT system inside a cartridge that is optically translucent and has multimodal fiducial markers for image fusion incorporated in its frame. While the immobilized mice were anesthetized with isoflurane, the holding device was transferred for ECG-gated high resolution CT (isotropic resolution 50um). During CT acquisition, iodine contrast was delivered at 65ul/min via tail vein. In vivo, we found strong fluorescent signal for uptake of nanoparticles as well as protease activity (p<0.05 WT vs apoE−/− mice), which was mapped to the proximal aorta on CT. Contrast enhanced CT visualized murine aortic plaques. Ex vivo fluorescence reflectance imaging and fluorescence microscopy corroborated in vivo findings and identified macrophages as target cells. FMT-CT imaging of inflammatory atherosclerosis provides quantifiable 3-dimensional maps of macrophage activity and visualizes murine aortic plaques, providing unprecedented insight into disease progression non-invasively.