The engineering of drug-encapsulated targeted nanoparticles (NPs) has the potential to revolutionize drug therapy. A major challenge for the smooth translation of targeted NPs to the clinic has been developing methods for the prediction and optimization of the NP surface composition, especially when targeting ligands (TL) of different chemical properties are involved in the NP self-assembly process. Here we investigated the self-assembly and properties of two different targeted NPs decorated with two widely used TLs that have different water solubilities, and developed methods to characterize and optimize NP surface composition. We synthesized two different biofunctional polymers composed of poly(lactide-co-glycolide)-b-polyethyleneglycol-RGD (PLGA-PEG-RGD, high water solubility TL) and PLGA-PEG-Folate (low water solubility TL). Targeted NPs with different ligand densities were prepared by mixing TL-conjugated polymers with non-conjugated PLGA-PEG at different ratios through nanoprecipitation. The NP surface composition was quantified and the results revealed two distinct nanoparticle assembly behaviors: for the case of PLGA-PEG-RGD, nearly all RGD molecules conjugated to the polymer were found to be on the surface of the NPs. In contrast, only ~20% of the folate from PLGA-PEG-Folate was present on the NP surface while the rest remained presumably buried in the PLGA NP core due to hydrophobic interactions of PLGA and folate. Finally, in vitro phagocytosis and cell targeting of NPs was investigated, from which a window of NP formulations exhibiting minimum uptake by macrophages and maximum uptake by targeted cells was determined. These results underscore the impact the ligand chemical properties have on the targeting capabilities of self-assembled targeted nanoparticles and provide an engineering strategy for improving their targeting specificity.

Interleukin-1 receptor (IL1R)-associated kinase 4 (IRAK4) is a central regulator of innate immune signaling, controlling IL1R and Toll-like receptor (TLR)-mediated responses and containing both scaffolding and kinase activities. Humans deficient in IRAK4 activity have autosomal recessive primary immune deficiency (PID). Here, we characterized the molecular mechanism of dysfunction of two IRAK4 PID variants, G298D and the compound variant R12C (R12C/R391H/T458I). Using these variants and the kinase-inactive D329A variant to delineate the contributions of IRAK4's scaffolding and kinase activities to IL1R signaling, we found that the G298D variant is kinase-inactive and expressed at extremely low levels, acting functionally as a null mutation. The R12C compound variant possessed WT kinase activity, but could not interact with myeloid differentiation primary response 88 (MyD88) and IRAK1, causing impairment of IL-1-induced signaling and cytokine production. Quantitation of IL-1 signaling in IRAK4-deficient cells complemented with either WT or the R12C or D329A variant indicated that the loss of MyD88 interaction had a greater impact on IL-1-induced signaling and cytokine expression than the loss of IRAK4 kinase activity. Importantly, kinase-inactive IRAK4 exhibited a greater association with MyD88 and a weaker association with IRAK1 in IRAK4-deficient cells expressing kinase-inactive IRAK4 and in primary cells treated with a selective IRAK4 inhibitor. Loss of IRAK4 kinase activity only partially inhibited IL-1-induced cytokine and NF-κB signaling. Therefore, the IRAK4-MyD88 scaffolding function is essential for IL-1 signaling, but IRAK4 kinase activity can control IL-1 signal strength by modulating the association of IRAK4, MyD88, and IRAK1.

Protein/ice interactions are investigated by a novel method based on measuring the characteristic features of X-ray diffraction (XRD) patterns of hexagonal ice (Ih). Aqueous solutions of four proteins and other solutes are studied using high-resolution synchrotron XRD. Two pharmaceutical proteins, recombinant human albumin and monoclonal antibody (both at 100 mg/mL), have a pronounced effect on the properties of ice crystals, reducing the size of the Ih crystalline domains and increasing the microstrain. Lysozyme (100 mg/ mL) and an antifreeze protein (1 mg/mL) have much weaker impact on Ih. Neither of the proteins studied exhibit preferred interactions with specific crystalline faces of Ih. It is proposed that the pharmaceutical proteins interact with ice crystals indirectly by accumulating in the quasi-liquid layer next to ice crystallization front, rather than directly, via a sorption on ice crystals. This is the first report, to the best of our knowledge, of major difference in the protein/ice interaction between non-antifreeze proteins. Another important finding is a detection of a second (minor) population of ice crystals, which is tentatively identified as a high-pressure form of ice, possibly IceIII or IceIX. This finding highlights a potential role of mechanical stresses in freeze-induced destabilization of proteins.

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