Targeted-nanotherapeutics (NP) have largely failed to translate clinically in showcasing improved disease-specific localization and associated drug delivery efficacy, stemming from a lack of insight into the influence, the type of targeting ligand/ligand density has on nano-bio interactions such as the serum protein corona formation that dictate NP fate in vivo [1]. We have previously developed a therapeutic nano-formulation with decreased adhesivity to blood and non-specific tumor tissue components while maintaining a strong cell surface receptor-specific binding affinity (termed DART nanoparticles) [2]. Recently, we demonstrated in vivo, the efficacy of paclitaxel-loaded DART nanoparticles directed to the cell surface receptor: fibroblast growth factor inducible 14 (Fn14), in primary as well as metastatic triplenegative breast cancer models [3]. In this present work, we investigated the impact of varying the Fn14- specific targeting moiety type- full-length monoclonal antibody (ITEM4 mAb) or fragment, antigen binding (ITEM4 Fab)- on DART NPs surface; On NP-Fn14 target-specificity and the subsequent cellular-uptake profiles by human glioma cell lines. We extensively examined the relationship between the targeting moiety type (ITEM4-mAb vs ITEM4-Fab) and the associated NP-ligand surface density on 1) Fn14 specific binding profiles and affinities 2) The resulting cellular-uptake rates on human glioma cell lines, and the influence of NP-serum protein corona formation on these processes

Background: Renal cyst formation in ADPKD requires altered cell and tubular morphology. Previously, we used inducible inactivation of Pkd2 in a 3D-tubuloid model to measure acute changes in tubuloid shape after Pkd2 loss and observed alterations in tubule morphology before changes in overall volume, arguing that morphological changes are among the first consequences of polycystin loss. We hypothesized a direct role for the polycystin complex in regulating the apical cytoskeleton protein, ezrin (EZR), to control cell and tubular morphology. Methods: We used clonal cell lines derived from the kidneys of Pkd1 or Pkd2 Pax8rtTA TetOCre mice crossed with the SV40 immorto-mouse: Pkd1-iKO (#312) and Pkd2-iKO (#125). Pkd1/2 can be efficiently deleted with addition of Doxycyline (DOX) to the cell media. Results: We observed a strong correlation between the loss of Pkd1/2 and increased apical cell area, as defined by ZO1, in 2D Pkd1/2-iKO cells. The acute loss of Pkd1/2 also resulted in decreased ezrin abundance and altered localization. Although Pkd1/2 deletion resulted in decreased ezrin we found upregulation of Ezr mRNA in both the DOX treated Pkd2-iKO cells and in human ADPKD cystic tissue, leading us to hypothesize that PC1/2 regulates ezrin protein more directly. Immunoprecipitation experiments of HEK293 cells transfected with ezrin, Myc-PC2 or Flag-PC1 showed both PC1 and PC2 successfully pulled down ezrin suggesting a protein interaction. Interestingly, the pull down efficiency was greater with the full length PC1 than the CTF- PC1 fragment. To confirm an endogenous interaction between ezrin and PC1 we isolated primary renal epithelial cells from a transgenic Pkd1-HA mouse and found again HA-PC1 successfully pulled down ezrin. Mechanistically, ezrin activity is regulated by cystine proteases calpain 1 and 2. We observed a substantial increase in the N-terminal 55KDa ezrin cleavage product after acute Pkd2 loss in the DOX treated Pkd2- iKO cells as well in human APDKD cystic tissue. Chemical inhibition of either calpain 1 or calpain 2 led to increased ezrin cleavage in control cells mimicking Pkd2 loss. However, inhibition of both calpain 1 and 2 reduced ezrin cleavage in DOX treated Pkd2-iKO cells to levels comparable to control cells. Conclusion: The polycystin proteins regulate ezrin function and cleavage to control renal cell and tubular morphology.

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