In vivo coherent anti-Stokes Raman scattering microscopy reveals vitamin A distribution in the liver

Here we present a microscope setup for coherent anti-Stokes Raman scattering (CARS) imaging, devised to specifically address the challenges of in vivo experiments. We exemplify its capabilities by demonstrating how CARS microscopy can be used to identify vitamin A (VA) accumulations in the liver of a living mouse, marking the positions of hepatic stellate cells (HSCs). HSCs are the main source of extracellular matrix protein after hepatic injury and are therefore the main target of novel nanomedical strategies in the development of a treatment for liver fibrosis. Their role in the VA metabolism makes them an ideal target for a CARS-based approach as they store most of the body's VA, a class of compounds sharing a retinyl group as a structural motive, a moiety that is well known for its exceptionally high Raman cross section of the C=C stretching vibration of the conjugated backbone

Stealth Effect of Short Polyoxazolines in Graft Copolymers: Minor Changes of Backbone End Group Determine Liver Cell-Type Specificity

Dye-loaded micelles of 10 nm diameter formed from amphiphilic graft copolymers composed of a hydrophobic poly(methyl methacrylate) backbone and hydrophilic poly(2-ethyl-2-oxazoline) side chains with a degree of polymerization of 15 were investigated concerning their cellular interaction and uptake in vitro as well as their interaction with local and circulating cells of the reticuloendothelial system in the liver by intravital microscopy. Despite the high molar mass of the individual macromolecules (Mn ≈ 20 kg mol-1), backbone end group modification by attachment of a hydrophilic anionic fluorescent probe strongly affected the in vivo performance. To understand these effects, the end group was additionally modified by the attachment of four methacrylic acid repeating units. Although various micelles appeared similar in dynamic light scattering and cryo-transmission electron microscopy, changes in the micelles were evident from principal component analysis of the Raman spectra. Whereas an efficient stealth effect was found for micelles formed from polymers with anionically charged or thiol end groups, a hydrophobic end group altered the micelles' structure sufficiently to adapt cell-type specificity and stealth properties in the liver. © 2021 The Authors. Published by American Chemical Society

Tuning the corona-core ratio of polyplex micelles for selective oligonucleotide delivery to hepatocytes or hepatic immune cells

Targeted delivery of oligonucleotides or small molecular drugs to hepatocytes, the liver's parenchymal cells, is challenging without targeting moiety due to the highly efficient mononuclear phagocyte system (MPS) of the liver. The MPS comprises Kupffer cells and specialized sinusoidal endothelial cells, efficiently clearing nanocarriers regardless of their size and surface properties. Physiologically, this non-parenchymal shield protects hepatocytes; however, these local barriers must be overcome for drug delivery. Nanocarrier structural properties strongly influence tissue penetration, in vivo pharmacokinetics, and biodistribution profile. Here we demonstrate the in vivo biodistribution of polyplex micelles formed by polyion complexation of short interfering (si)RNA with modified poly(ethylene glycol)-block-poly(allyl glycidyl ether) (PEG-b-PAGE) diblock copolymer that carries amino moieties in the side chain. The ratio between PEG corona and siRNA complexed PAGE core of polyplex micelles was chemically varied by altering the degree of polymerization of PAGE. Applying Raman-spectroscopy and dynamic in silico modeling on the polyplex micelles, we determined the corona-core ratio (CCR) and visualized the possible micellar structure with varying CCR. The results for this model system reveal that polyplex micelles with higher CCR, i.e., better PEG coverage, exclusively accumulate and thus allow passive cell-type-specific targeting towards hepatocytes, overcoming the macrophage-rich reticuloendothelial barrier of the liver

Characterization of a Library of Vitamin A-Functionalized Polymethacrylate-Based Nanoparticles for siRNA Delivery

A 60-membered library of vitamin A-functionalized P(MMA-stat-DMAEMA)-b-PPEGMA block copolymers was synthesized by RAFT polymerization. Subsequently, retinoic acid was coupled to hydroxyl groups present in the hydrophilic PPEGMA block. The polymers were investigated for their ability to encapsulate ribonucleic acids through nanoparticle (NP) formulation using the emulsion/solvent evaporation method. The localization of vitamin A in surface-near regions of the NPs was indicated by surface enhanced Raman spectroscopy, and the interaction of the NPs with a retinol binding protein was investigated by analytical ultracentrifugation. The systematic analysis of the NP library in terms of the encapsulation efficiency of the ribonucleic acids, the toxicity of the NPs, and the cellular uptake helped identifying suitable candidates for cellular internalization studies. The cell uptake was investigated by flow cytometry and fluorescence microscopy and reveals structure dependent uptake behavior of the examined particles. </p