Dissecting the Carbohydrate Specificity of the Anti-HIV‑1 2G12 Antibody by Single-Molecule Force Spectroscopy

Broadly neutralizing anti-HIV-1 monoclonal antibody 2G12 exclusively targets a conserved cluster of high-mannose oligosaccharides present on outer viral envelope glycoprotein gp120. This characteristic makes the otherwise immunogenically “silent” glycan shield of gp120 a tempting target for drug and vaccine design. However, immune responses against carbohydrate-based mimics of gp120 have failed to provide immunization against HIV-1 infection, highlighting the need to understand the molecular events that determine immunogenicity better. In this work, the unbinding kinetics of the gp120–2G12 (<i>k</i>₀ = 0.002 ± 0.09 s^–1, <i>x</i>^⧧ = 1.5 ± 1.2 nm), Man₄–2G12 (<i>k</i>₀ = 0.35 ± 0.32 s^–1, <i>x</i>^⧧ = 0.6 ± 0.2 nm), and Man₅–2G12 interactions were measured by single-molecule force spectroscopy. To our knowledge, this is the first single-molecule study aimed at dissecting the carbohydrate–antibody recognition of the gp120–2G12 interaction. We were able to confirm crystallographic models that show both the binding of the linear Man₄ arm to 2G12 and also the multivalent gp120 glycan binding to 2G12. These results demonstrate that single-molecule force spectroscopy can be successfully used to dissect the molecular mechanisms underlying immunity

⁶⁸Ga-Labeled Gold Glyconanoparticles for Exploring Blood–Brain Barrier Permeability: Preparation, Biodistribution Studies, and Improved Brain Uptake via Neuropeptide Conjugation

New tools and techniques to improve brain visualization and assess drug permeability across the blood–brain barrier (BBB) are critically needed. Positron emission tomography (PET) is a highly sensitive, noninvasive technique that allows the evaluation of the BBB permeability under normal and disease-state conditions. In this work, we have developed the synthesis of novel water-soluble and biocompatible glucose-coated gold nanoparticles (GNPs) carrying BBB-permeable neuropeptides and a chelator of the positron emitter ⁶⁸Ga as a PET reporter for in vivo tracking biodistribution. The small GNPs (2 nm) are stabilized and solubilized by a glucose conjugate. A NOTA ligand is the chelating agent for the ⁶⁸Ga, and two related opioid peptides are used as targeting ligands for improving BBB crossing. The radioactive labeling of the GNPs is completed in 30 min at 70 °C followed by purification via centrifugal filtration. As a proof of principle, a biodistribution study in rats is performed for the different ⁶⁸Ga-GNPs. The accumulation of radioactivity in different organs after intravenous administration is measured by whole body PET imaging and gamma counter measurements of selected organs. The biodistribution of the ⁶⁸Ga-GNPs varies depending on the ligands, as GNPs with the same gold core size show different distribution profiles. One of the targeted ⁶⁸Ga-GNPs improves BBB crossing near 3-fold (0.020 ± 0.0050% ID/g) compared to nontargeted GNPs (0.0073 ± 0.0024% ID/g) as measured by dissection and tissue counting

Residual CTAB Ligands as Mass Spectrometry Labels to Monitor Cellular Uptake of Au Nanorods

Gold nanorods have numerous applications in biomedical research, including diagnostics, bioimaging, and photothermal therapy. Even though surfactant removal and surface conjugation with antifouling molecules such as polyethylene glycol (PEG) are required to minimize nonspecific protein binding and cell uptake, the reliable characterization of these processes remains challenging. We propose here the use of laser desorption/ionization mass spectrometry (LDI-MS) to study the ligand exchange efficiency of cetyltrimethylammonium bromide (CTAB)-coated nanorods with different PEG grafting densities and to characterize nanorod internalization in cells. Application of LDI-MS analysis shows that residual CTAB consistently remains adsorbed on PEG-capped Au nanorods. Interestingly, such residual CTAB can be exploited as a mass barcode to discern the presence of nanorods in complex fluids and in vitro cellular systems, even at very low concentrations

Cellular Uptake of Gold Nanoparticles Triggered by Host–Guest Interactions

We describe an approach to regulate the cellular uptake of small gold nanoparticles using supramolecular chemistry. The strategy relies on the functionalization of AuNPs with negatively charged pyranines, which largely hamper their penetration in cells. Cellular uptake can be activated <i>in situ</i> through the addition of cationic covalent cages that specifically recognize the fluorescent pyranine dyes and counterbalance the negative charges. The high selectivity and reversibility of the host–guest recognition activates cellular uptake, even in protein-rich biological media, as well as its regulation by rational addition of either cage or pyranine