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Nanoparticles (NPs) are commonly modified with tumor-targeting moieties that recognize proteins overexpressed on the extracellular membrane to increase their specific interaction with target cells. Nanobodies (Nbs), the variable domain of heavy chain-only antibodies, are a robust targeting ligand due to their small size, superior stability, and strong binding affinity. For the clinical translation of targeted Nb-NPs, it is essential to understand how the number of Nbs per NP impacts the receptor recognition on cells. To study this, Nbs targeting the hepatocyte growth factor receptor (MET-Nbs) were conjugated to PEGylated liposomes at a density from 20 to 800 per liposome and their targeting efficiency was evaluated in vitro. MET-targeted liposomes (MET-TLs) associated more profoundly with MET-expressing cells than non-targeted liposomes (NTLs). MET-TLs with approximately 150–300 Nbs per liposome exhibited the highest association and specificity towards MET-expressing cells and retained their targeting capacity when pre-incubated with proteins from different sources. Furthermore, a MET-Nb density above 300 Nbs per liposome increased the interaction of MET-TLs with phagocytic cells by 2-fold in ex vivo human blood compared to NTLs. Overall, this study demonstrates that adjusting the MET-Nb density can increase the specificity of NPs towards their intended cellular target and reduce NP interaction with phagocytic cells. [ABSTRACT FROM AUTHOR]

Nanobody-targeted photodynamic therapy (NB-PDT) has been recently developed as a more tumor-selective approach rather than conventional photodynamic therapy (PDT). NB-PDT uses nanobodies that bind to tumor cells with high affinity, to selectively deliver a photosensitizer, i.e., a chemical which becomes cytotoxic when excited with light of a particular wavelength. Conventional PDT has been reported to be able to induce immunogenic cell death, characterized by the exposure/release of damage-associated molecular patterns (DAMPs) from dying cells, which can lead to antitumor immunity. We explored this aspect in the context of NB-PDT, targeting the epidermal growth factor receptor (EGFR), using high and moderate EGFR-expressing cells. Here we report that, after NB-PDT, the cytoplasmic DAMP HSP70 was detected on the cell membrane of tumor cells and the nuclear DAMP HMGB1 was found in the cell cytoplasm. Furthermore, it was shown that NB-PDT induced the release of the DAMPs HSP70 and ATP, as well as the pro- inflammatory cytokines IL- 1β and IL-6. Conditioned medium from high EGFR-expressing tumor cells treated with NB-PDT led to the maturation of human dendritic cells, as indicated by the upregulation of CD86 and MHC II on their cell surface, and the increased release of IL-12p40 and IL-1β. Subsequently, these dendritic cells induced CD4+ T cell proliferation, accompanied by IFNγ release. Altogether, the initial steps reported here point towards the potential of NB-PDT to stimulate the immune system, thus giving this selective-local therapy a systemic reach. [ABSTRACT FROM AUTHOR]