Crosslink-dependent pulmonary toxicity of pNIPAM-AAc microgels

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Nicosia, John Michael
Lam, Wilbur A.
Barker, Thomas H.
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Microgels, also referred to as nanogels, are hydrophilic polymer networks synthesized into micro- or nano-sized hydrogel particles. Due to their relative ease of synthesis and highly tunable properties, they have been explored extensively for a range of biomedical applications, including therapeutic delivery, imaging, and biosensors. Yet despite decades of research, only a handful of microgel-based technologies have reached clinical trials. Primary barriers to clinical translation of microgels include rapid clearance from circulation and toxicity or off-target effects. While there have been many advancements in microgel research, there still exists a lack of understanding of the factors that drive microgel behavior in vivo To address this gap in knowledge, this work uses poly(N-isopropylacrylamide)-co-acrylic acid (pNIPAM-AAc) microgels as a case study in how microgel crosslink density affects mechanical properties of microgels in physiologic settings, and correspondingly influences their behavior in vivo. The central hypothesis of this work is that increased crosslinking leads to shorter blood circulation time of microgels, and enhanced margination to the walls of blood vessels. Microgel characteristics were evaluated with a combination of single microgel atomic force microscopy (AFM) nanoindentation techniques along with light scattering and rheological analysis of microgel suspensions. A variety of microfluidic systems were used to investigate microgel behavior in whole blood, while in vivo testing was carried out to determine biodistribution, clearance time, and potential toxicity. Increased crosslinking had minimal effect on clearance time and margination of pNIPAM-AAc microgels, though crosslinking was associated with longer retention time in the kidneys. During biodistribution studies, it was observed that some mice who received crosslinked microgels showed signs of significant toxicity. Follow-up in vitro and in vivo studies revealed that crosslinked microgels have a greater tendency to form large aggregates in blood that can occlude lung microvasculature, though this does not lead to an overall inflammatory response. Instead, it is likely that crosslinked microgels occasionally form especially large aggregates that result in rare catastrophic events similar to a pulmonary embolism. Further exploration of this phenomenon revealed that aggregate formation is driven by hydrophobic interactions and exacerbated by the binding of plasma proteins such as albumin. Rational design of microgel-based therapies should utilize stably hydrophilic polymers to minimize protein binding and reduce the risk of aggregate formation in blood.
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