Adhesion of Blood Clots Can Be Enhanced When Copolymerized with a Macromer That Is Crosslinked by Coagulation Factor XIIIa.

The adhesion of blood clots to blood vessels, such as through the adhesion of fibrin, is essential in hemostasis. While numerous strategies for initiating clot formation and preventing clot lysis are being developed to create improved hemostatic agents, strategies for enhancing clot adhesion have not been widely explored. Here, we show that adhesion of blood clots can be increased by adding a previously characterized synthetic polymer that is crosslinked by coagulation factor XIIIa during clotting. Addition of the polymer to normal plasma increased the adhesive strength of clots by 2-fold. It also recovered the adhesive strength of nonadhesive fibrinogen-deficient whole blood clots from <0.06 kPa to 1.9 ± 0.14 kPa, which is similar to the adhesive strength of a fibrinogen-rich clot (1.8 ± 0.64 kPa). The polymer also enabled plasma clots to remain adhered under fibrinolytic conditions. By demonstrating that the adhesive strength of clots can be increased with a synthetic material, this provides a potential strategy for creating advanced hemostatic materials, such as treatments for fibrinogen deficiency in trauma-induced coagulopathy.

Determination of the interior pH of lipid nanoparticles using a pH-sensitive fluorescent dye-based DNA probe.

Lipid nanoparticles (LNPs) containing ionizable cationic lipids are proven delivery systems for therapeutic nucleic acids, such as small interfering RNA (siRNA). It is important to understand the relationship between the interior pH of LNPs and the pH of the external environment to understand LNP formulation and function. Here, we developed a simple and rapid approach for determining the pH of the LNP core using a pH-sensitive fluorescent dye-based DNA probe. LNP siRNA systems containing pH-responsive DNA probes (LNP-siRNA&DNA) were generated by rapid mixing of lipids in ethanol and pH 4 aqueous buffer containing siRNA and DNA probes. We demonstrated that DNA probes were readily encapsulated in LNP systems and were sequestered into an environment at a high concentration as evidenced by an inter-probe FRET signal. It was shown that the pH of LNP encapsulated probes closely follows the pH increase or decrease of the external environment. This indicates that the clinically approved LNP RNA systems with similar lipid compositions (e.g., Onpattro and Comirnaty) are highly permeable to protons and that the pH of the interior environment closely mirrors the external environment. The pH-dependent response of the probe in LNPs was also confirmed under buffer conditions at various pHs. Furthermore, we showed that the pH-sensitive DNA probe can be incorporated into LNP systems at levels that allow the pH response to be monitored at a single LNP level using convex lens-induced confinement (CLiC) confocal microscopy. Direct visualization of the internal pH of single particles with the fluorescent DNA probe was achieved by CLiC for LNP-siRNA&DNA systems formulated under both high and normal ionic strength conditions.

Anionic Lipid Nanoparticles Preferentially Deliver mRNA to the Hepatic Reticuloendothelial System.

Lipid nanoparticles (LNPs) are the leading nonviral technologies for the delivery of exogenous RNA to target cells in vivo. As systemic delivery platforms, these technologies are exemplified by Onpattro, an approved LNP-based RNA interference therapy, administered intravenously and targeted to parenchymal liver cells. The discovery of systemically administered LNP technologies capable of preferential RNA delivery beyond hepatocytes has, however, proven more challenging. Here, preceded by comprehensive mechanistic understanding of in vivo nanoparticle biodistribution and bodily clearance, an LNP-based messenger RNA (mRNA) delivery platform is rationally designed to preferentially target the hepatic reticuloendothelial system (RES). Evaluated in embryonic zebrafish, validated in mice, and directly compared to LNP-mRNA systems based on the lipid composition of Onpattro, RES-targeted LNPs significantly enhance mRNA expression both globally within the liver and specifically within hepatic RES cell types. Hepatic RES targeting requires just a single lipid change within the formulation of Onpattro to switch LNP surface charge from neutral to anionic. This technology not only provides new opportunities to treat liver-specific and systemic diseases in which RES cell types play a key role but, more importantly, exemplifies that rational design of advanced RNA therapies must be preceded by a robust understanding of the dominant nano-biointeractions involved.

A biochemical network can control formation of a synthetic material by sensing numerous specific stimuli.

Developing bio-compatible smart materials that assemble in response to environmental cues requires strategies that can discriminate multiple specific stimuli in a complex milieu. Synthetic materials have yet to achieve this level of sensitivity, which would emulate the highly evolved and tailored reaction networks of complex biological systems. Here we show that the output of a naturally occurring network can be replaced with a synthetic material. Exploiting the blood coagulation system as an exquisite biological sensor, the fibrin clot end-product was replaced with a synthetic material under the biological control of a precisely regulated cross-linking enzyme. The functions of the coagulation network remained intact when the material was incorporated. Clot-like polymerization was induced in indirect response to distinct small molecules, phospholipids, enzymes, cells, viruses, an inorganic solid, a polyphenol, a polysaccharide, and a membrane protein. This strategy demonstrates for the first time that an existing stimulus-responsive biological network can be used to control the formation of a synthetic material by diverse classes of physiological triggers.