Targeting Ligand Independent Tropism of siRNA‐LNP by Small Molecules for Directed Therapy of Liver or Myeloid Immune Cells

Abstract

Hepatic clearance of lipid nanoparticles (LNP) with encapsulated nucleic acids restricts their therapeutic applicability. Therefore, tools for regulating hepatic clearance are of high interest for nucleic acid delivery. To this end, this work employs wild‐type (WT) and low‐density lipoprotein receptor (Ldlr)−/− mice pretreated with either a leukotriene B4 receptor inhibitor (BLT1i) or a high‐density lipoprotein receptor inhibitor (HDLRi) prior to the injection of siRNA‐LNP. This work is able to demonstrate significantly increased hepatic uptake of siRNA‐LNP by the BLT1i in Ldlr−/− mice by in vivo imaging and discover an induction of specific uptake‐related proteins. Irrespective of the inhibitors and Ldlr deficiency, the siRNA‐LNP induced RNA‐binding and transport‐related proteins in liver, including haptoglobin (HP) that is also identified as most upregulated serum protein. This work observes a downregulation of proteins functioning in hepatic detoxification and of serum opsonins. Most strikingly, the HDLRi reduces hepatic uptake and increases siRNA accumulation in spleen and myeloid immune cells of blood and liver. RNA sequencing demonstrates leukocyte recruitment by the siRNA‐LNP and the HDLRi through induction of chemokine ligands in liver tissue. The data provide insights into key mechanisms of siRNA‐LNP biodistribution and indicate that the HDLRi has potential for extrahepatic and leukocyte targeting.

Macrophage‐like rapid uptake and toxicity of tattoo ink in human monocytes

“…The first method employed DLS settings that we have used in earlier studies. Routinely we use DLS as it is done in the first method where we characterize for instance lipid nanoparticles for siRNA, at a DLS angle of 173.5° [15]. This method revealed that at a concentration of 1:10 diluted in phosphate‐buffered saline (PBS), the ink particles sized 1080 nm, but when they were diluted 1:100 in PBS, the size increased to 5615 nm (Table S1).…”

“…We suggest that huge amounts of the ink are stored in vesicles of monocytes and macrophages and that this is owed to the large aggregations of the ink. Clinically usable nanoparticles such as lipid nanoparticles for siRNA [15], or liposomal carriers [20], do not affect the granularity of immune cells, which is likely based on the fact that these nano‐sized particles do not lead to large intracellular inclusions.…”

“…After homogenization, cells were digested with collagenase type IV. More details can be found in previous studies where the method was used to isolate cells from liver [15]. Cells from the skin models or cells from the monocultures were collected and homogenized.…”

A dynamic bias in chromatin protein deposition at G-quadruplex sites

“…The labeled nucleosomes were enriched by Dynabeads TM Streptavidin T1 following the manufacturer's instruction. Protein levels were determined with discovery-based proteomics (using label-free quantification) for relative protein concentrations (28). Briefly, in-gel digestion was performed on 40 µL of the proteins that were eluted from the beads in 1X LDS loading buffer (Thermo scientific) using trypsin (150 ng sequencing grade modified trypsin V5111; Promega) after reduction with 10 mmol/L dithiothreitol and alkylation with 55 mmol/L iodoacetamide proteins (29).…”

Just Keep Rolling?—An Encompassing Review towards Accelerated Vaccine Product Life Cycles

“…Active targeting can be executed with LNPs containing a target-specific ligand in the formulation, whereas passive targeting can be executed without targeting moiety [ 25 ]. Targeting moieties for active targeting mechanisms include antibodies [ 26 ], aptamers [ 27 ], small molecules [ 28 ] and proteins or peptides [ 29 ]. Passive targeting (EPR-effect) is influenced by factors such as the size and charge of the LNP, which depend on the molar composition of the different types of lipids used in the formulation [ 30 ].…”