Squaric Ester‐Based Nanogels Induce No Distinct Protein Corona but Entrap Plasma Proteins into their Porous Hydrogel Network

Abstract

After intravenous administration of nanocarriers, plasma proteins may rapidly adsorb onto their surfaces. This process hampers the prediction of the nanocarriers' pharmacokinetics as it determines their physiological identity in a complex biological environment. Toward clinical translation it is therefore an essential prerequisite to investigate the nanocarriers' interaction with plasma proteins. Here, this work evaluates a highly "PEGylated" squaric ester-based nanogel with inherent prolonged blood circulation properties. After incubation with human blood plasma, the nanogels are isolated by asymmetrical flow-field flow fractionation. Multiangle light scattering measurements confirm the absence of significant size increases as well as aggregation upon plasma incubation. However, proteomic analyses by gel electrophoresis find minor absolute amounts of proteins (3 wt%), whereas label-free liquid chromatography mass spectrometry identify 65 enriched proteins. Interestingly, the relative abundance of these proteins is almost similar to their proportion in pure native plasma. Due to the nanogels' hydrated and porous network morphology, it is concluded that the detected proteins rather result from passive diffusion into the nanogel network than from specific interactions at the plasma particle interface. Consequently, these results do not indicate a classical surface protein corona but rather reflect the highly outer and inner stealth-like behavior of the porous hydrogel network.

Post‐Polymerization Modifications to Prepare Biomedical Nanocarriers with Varying Internal Structures, their Properties and Impact on Protein Corona Formation

“…Recent experiments demonstrated, however, that such nano(hydro)gels absorb only very little plasma proteins when they are made from highly polar and stealth polymers. [87] For the squarogel system with short PEG chains inside the core, their tendency to adsorb plasma proteins is comparably as low as what we observed also for block copolymer micelles (Figure 5 and Figure 6). [56] However, there was a significant difference in the class of the plasma proteins that were found after separation by AF4 and subsequent proteomic analysis by mass spectrometry.…”

“…This explains why there was no preferential fraction of proteins found by mass spectrometry which would suggest a favorable polymer-protein interaction (but more or less conclude a "stealthness even inside the nanogel"). [87] In contrast, the proteins identified by mass spectrometry after plasma incubation with block-copolymer micelles [56,117] could mostly be identified as rare proteins related to classical cholesterol/ triacylglyceride transporter proteins in the body (e.g., apo lipoproteins etc.). Such proteins probably provide favorable interaction sides with the partially less stabilized hydrophilic/ hydrophobic interface and are, therefore, selectively recruited by the micelles irreversibly (Figure 5 and Figure 6).…”

“…The composition of proteins associated with the squarogels corresponded mostly with their natural abundancy in the same blood plasma applied during incubation. [87] Having a closer look, it seemed that smaller proteins were slightly preferred and large plasma proteins got rather excluded. We hypothesize that this is due to size discrimination of the core-crosslinked network in the interior of the nano(hydro)gels.…”

“…For that purpose, we more recently introduced the squaric ester amide technology as an attractive alternative to performing a nano(hydro)gels synthesis in water affording so-called squarogels. [73,85,87,88] Due to the high sensitivity to amines but hydrolytic resistance of the squaric ester amides in water, respec-Scheme 1. Reaction scheme for the preparation of poly(norbornene)-nano(hydro)gels.…”

“…The alendronate-loaded squarogels promoted an improved delivery of the bisphosphonate to immunosuppressive macrophages in fibrotic livers and thereby repolarized their phenotype in order to prevent liver fibrosis disease progression. [73,85,87,88] As a result of all these different studies, one should take into account that a large variety of nanoparticles with different structures can be prepared from block copolymers bearing amine reactive blocks: Beyond polymeric micelles with a hydrophobic core (Figure 1), polymeric nano(hydro)gels seem to be very robustly reproduced by this strategy (Figure 2). Although the inner part can chemically differ from each other, upon core-crosslinking and hydrophilization it can provide nano(hydro)gels of comparable inner and outer hydrophilicity as well as inner mechanical property.…”