Poster X20 - Impact of protein corona on the molecular specificity and cellular uptake of decreased nonspecific adhesivity, receptor-targeted (DART) nanoparticles for clinical translation

Introduction: Nanoparticle-based drug delivery, exemplified by notable successes of (Doxil), irinotecan (ONIVYDE)[1] and nano-paclitaxel (Abraxane) [2], has enhanced cancer therapy by reducing drug toxicity and increasing drug circulation time. However, these nanotherapeutics often show poor tumor accumulation/retention [3, 4], and functionalizing nanoparticles with molecular target-specific entities such as monoclonal antibodies, or smaller fragments has emerged as an approach to improve tissue/cell-specific drug delivery. Currently, the successful clinical translation of targeted nanotherapeutics hinges on elucidating the effect of the type of targeting entity/density on the nano-bio interactions like serum protein corona formation that govern nanoparticle fate in vivo [5].
We have developed a nano-formulation with decreased adhesivity to blood and nonspecific tumor tissue components while maintaining a strong cell surface receptor-specific binding affinity (termed DART nanoparticles) [6]. 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 triple-negative breast cancer models [7]. 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 and triple-negative breast cancer (TNBC) cell lines and the influence of NP-serum protein corona formation on these processes. Finally, we also show, that despite similar accumulation in brains harboring TNBC tumors, Fn14-targeted DARTs exhibit significant and specific association with Fn14-positive TNBC cells compared to nontargeted NPs or Abraxane.

Materials and

Methods:

Methods: We formulated poly(lactic-co-glycolic acid)-poly(ethylene glycol) based (PLGA-PEG) nanoparticles with maleimide functionalization via standard oil in water emulsion methods and decorated the NP surfaces with varying surface densities of either Fn14 directed monoclonal antibody (ITEM4 mAb) or the fragment antigen binding region of ITEM4 (ITEM4 Fab) using thiol-maleimide based bioconjugation techniques thus creating a library of DART-ITEM4 mAb or DART-ITEM4 Fab NPs. This DART NP library was physiochemically characterized via dynamic light scattering techniques and peptide assays to quantify respective ligand surface densities. Next, we used surface plasmon-based assays to probe the Fn14-specific binding affinities of these libraries, alongside flow cytometry-based methods and total internal reflection fluorescence (TIRF) microscopy-based methods to dissect the cellular uptake profiles as well as temporal uptake rates of DART-ITEM4 mAb and DART-ITEM4 Fab NPs under the presence and absence of serum protein corona formation. Finally, NPs were injected into brain tumor-harboring mice and after 24h the entire left hemisphere of the brain harboring the tumor was isolated, processed, and stained with immunophenotypic markers for flow cytometric analysis. A standardized gating strategy was used to identify tumor cells (for 231-TD, CD45–/GFP+; for 66.1-luc, CD45–/luciferase+), monocytes (CD45+CD11b+Ly6C+Ly6G−), neutrophils (CD45+CD11b+Ly6C-Ly6G+), tumor-associated macrophages (CD45+CD11b+Ly6C-Ly6G−), and putative lymphocytes (CD45+CD11b−).

Results, Conclusions, and Discussions:

Results: SPR assays revealed an optimal NP surface ligand type and density (ITEM4 mAb/ < 13 mAbs/NP) as optimal for high Fn14 specific binding and it’s retention under the influence of serum protein corona formation. Flow cytometry analysis showcased dose, time and Fn14 expression level-dependent cellular uptake by human glioma and TNBC cell lines. Furthermore, TIRF microscopy and SPR analysis indicated that serum protein corona formation dramatically reduces total NP cellular uptake regardless of targeting but results in significantly increased specificity of the Fn14-targeted NPs conjugated with ITEM4 mAb. Lastly, we found that Fn14-targeted NPs are predominantly internalized via clathrin-mediated endocytosis and traffic via the endo-lysosomal pathway. Flow cytometry analysis of the total Cy5 signal of each population revealed significant, specific targeting of Fn14-positive tumor cells with Cy5-PP-IT4 NP treatment in both TNBC brain metastasis models (Figure 1) and significantly less Cy5-PP-IgG NP and Cy5-Abraxane accumulation in these same cells. Cy5-PP-IT4 NPs exhibited 6.2- and 2.9-fold more accumulation than Cy5-PP-IgG NP within the tumor cell compartment of 231-TD-luc and 66.1-luc brain tumors, respectively.

Acknowledgements (Optional): Acknowledgments: This work was supported, in part, by the National Institutes of Health (R37 CA212617 and R01 NS107813) and the United States Department of Veterans Affairs (I01 BX004908).