Nanobody-conjugated nanoparticles for photodynamic therapy: steps towards a targeted therapy for cholangiocarcinoma

The aim of this work was to develop stable nanoparticles (NPs) for the systemic delivery of both hydrophobic and hydrophilic phthalocyanines that could improve specific accumulation of these photosensitizers (PS) in the tumor for targeted photodynamic therapy (PDT). Moreover, nanobodies with affinity to overexpressed membrane proteins were produced and conjugated to the surface of the NPs to facilitate their specific interaction with and internalization by cancer cells. The multivalency of these NPs in combination with a high number of target proteins on cancer cells theoretically increases their binding probability, which in turn augments the number of PS molecules that are internalized, and ultimately the extent of phototoxicity upon photodynamic therapy. This strategy intends to improve the PDT efficacy and advance its application as standard cancer treatment for cholangiocarcioma (CCA).In this thesis, different nanocarriers (e.g., liposomes and micelles) were employed in an attempt to improve the stability of different phthalocyanine-based photosensitizers (PSs) in the bloodstream and improve the accumulation of the PS in the tumor. The nanoparticles (NPs) with the highest retention of the PS when incubated with human plasma were selected using asymmetric flow-field flow fractionation, a technique that could be applied to test other active pharmaceutical ingredients and be an integrative part of the characterization of NPs in the pharmaceutical industry. We have also shown that nanobody (Nb)-targeted nanocarriers are very effective in vitro at increasing uptake and specific cell death in high membrane protein-expressing cancer cells, including cholangiocarcinoma (CCA) cells. The first in vivo studies performed with a CCA xenograft mouse model revealed an efficient accumulation of tetrasulphonated aluminium phthalocyanine in the tumor of mice injected with target and non-targeted liposomes. However, the Nb-targeted liposomes did not increase the overall PS association with cancer cells. While the initial hypothesis of this thesis was not confirmed in vivo, the PDT efficacy of PS-loaded nanocarriers remains to be compared with standard PDT regimens. Moreover, the data presented here raised relevant questions for future research in PDT.