EGFR targeted nanobody functionalized polymeric micelles loaded with mTHPC for selective photodynamic therapy

Meta-tetra(hydroxyphenyl)chlorin (mTHPC) is one of the most potent second generation photosensitizers, clinically used for photodynamic therapy (PDT) of head and neck squamous cell carcinomas. However, improvements are still required concerning its present formulation (i.e. Foscan®, a solution of mTHPC in ethanol/propylene glycol (40:60 w/w)), as mTHPC has the tendency to aggregate in aqueous media, e.g. biological fluids, and it has limited tumor specificity. In the present study, polymeric micelles with three different diameters (17, 24 and 45 nm) based on benzyl-poly(ɛ-caprolactone)-b-poly(ethylene glycol) (PCLn-PEG; n=9, 15 or 23) were prepared with mTHPC loadings ranging from 0.5 to 10 wt% using a film hydration method as advanced nanoformulations for this photosensitizer. To favor the uptake of the micelles by cancer cells that over-express the epidermal growth factor receptor (EGFR), the micelles were decorated with an EGFR targeted nanobody (named EGa1) through maleimide-thiol chemistry. The enhanced binding of the EGFR targeted micelles at 4 ℃ to EGFR over-expressing A431 cells, compared to low EGFR expressing HeLa cells, confirmed the specificity of the micelles. In addition, an enhanced uptake of mTHPC loaded micelles by A431 cells was observed when these were decorated with EGa1 nanobody, compared to non-targeted micelles. Both binding and uptake of targeted micelles were blocked by an excess of free EGa1 nanobody, demonstrating that these processes occur through EGFR. In line with this, mTHPC-loaded in EGa1-conjugated PCL23-PEG (EGa1-P23) micelles demonstrated 4 times higher photocytotoxicity on A431 cells, compared to micelles lacking the nanobody. Importantly, EGa1-P23 micelles also showed selective PDT against A431 cells compared to the low EGFR-expressing HeLa cells. Finally, an in vivo pharmacokinetic study shows that after iv injection, mTHPC incorporated in the P23 micelles displayed prolonged blood circulation kinetics, compared to free mTHPC, independently of the presence of EGa1. Thus, these results make these micelles a promising nanomedicine formulation for selective therapy.