Folate-PEG Conjugates of a Far-Red Light-Activatable Paclitaxel Prodrug to Improve Selectivity toward Folate Receptor-Positive Cancer Cells

[Image: see text] We recently demonstrated the far-red light-activatable prodrug of paclitaxel (PTX), Pc-(L-PTX)(2). Upon illumination with a 690 nm laser, Pc-(L-PTX)(2) showed combinational cell killing from rapid photodynamic therapy damage by singlet oxygen, followed by sustained chemotherapy effects from locally released PTX. However, its high lipophilicity (log D(7.4) > 3.1) caused aggregation in aqueous solutions and has nonselectivity toward cancer cells. To solve these important problems, we prepared folic acid (FA)-conjugated and photoactivatable prodrugs of PTX with a polyethylene glycol (PEG) spacer of various chain lengths: FA-PEG(n)-Pc-L-PTX [n = 0 (0k, 5), ∼23 (1k, 7a), ∼45 (2k, 7b), ∼80 (3.5k, 7c), or ∼114 (5k, 7d)]. The PEGylated prodrugs 7a–d had a much improved hydrophilicity compared with the non-PEGylated prodrug, Pc-(L-PTX)(2). As the PEG length increased, the hydrophilicity of the prodrug increased (log D(7.4) values: 1.28, 0.09, −0.24, and −0.59 for 1k, 2k, 3.5k, and 5k PEG prodrugs, respectively). Fluorescence spectral data suggested that the PEGylated prodrugs had good solubility in the culture medium at lower concentrations (<1–2 μM), but showed fluorescence quenching due to limited solubility at higher concentrations (>2 μM). Dynamic light scattering indicated that all of the prodrugs formed nanosized particles in both phosphate-buffered saline and culture medium at a concentration of 5 μM. The PEG length affected both nonspecific and folate receptor (FR)-mediated uptake of the prodrugs. The enhanced cellular uptake was observed for the prodrugs with medium-sized PEGs (1k, 2k, or 3.5k) in FR-positive SKOV-3 cells, but not for the prodrugs with no PEG or with the longest PEG (5k), which suggests the optimal range of PEG length around 1k–3.5k for effective uptake of our prodrug system. Consistent with the cellular uptake pattern, medium-sized PEGylated prodrugs showed more potent phototoxic activity (IC(50s), ∼130 nM) than prodrugs with no PEG or the longest PEG (IC(50), ∼400 nM). In conclusion, we have developed far-red light-activatable prodrugs with improved water solubility and FR-targeting properties compared with the nontargeted prodrug.