Double-targeting using a TrkC ligand conjugated to dipyrrometheneboron difluoride (BODIPY) based photodynamic therapy (PDT) agent

Abstract

A molecule 1 (IY-IY-PDT) was designed to contain a fragment (IY-IY) that targets the TrkC receptor and a photosensitizer that acts as an agent for photodynamic therapy (PDT). Molecule 1 had submicromolar photocytotoxicities to cells that were engineered to stably express TrkC (NIH3T3-TrkC) or that naturally express high levels of TrkC (SY5Y neuroblastoma lines). Control experiments showed that 1 is not cytotoxic in the dark and has significantly less photocytotoxicity toward cells that do not express TrkC (NIH3T3-WT). Other controls featuring a similar agent 2 (YI-YI-PDT), which is identical and isomeric with 1 except that the targeting region is scrambled (a YI-YI motif, see text), showed that 1 is considerably more photocytotoxic than 2 on TrkC(+) cells. Imaging live TrkC(+) cells after treatment with a fluorescent agent 1 (IY-IY-PDT) proved that 1 permeates into TrkC(+) cells and is localized in the lysosomes. This observation indirectly indicates that agent 1 enters the cells via the TrkC receptor. Consistent with this, the dose-dependent PDT effects of 1 can be competitively reduced by the natural TrkC ligand, neurotrophin NT3.

Figure 1

a UV-Vis and b fluorescence spectra (excited at 450 nm) of 1 and 2 (both at 5.5 μM) in DMEM medium.

Figure 2

Cytotoxicities induced by IY-IY-PDT under light (red) and dark (blue) and scrambled negative control YI-YI- PDT under light (green) conditions for a cell line stably transfected with TrkC receptors. Throughout this paper, in the light experiments the cells were illuminated with a broad spectrum source, filtered to only deliver photons of >480 nm wavelength, at a flux of approximately 12.2 mW/cm² for 10 mins. Error bars were based on 3 runs.

Figure 3

Dose-dependent reduction of IY-IY-PDT photoinduced cytotoxicity (red) in competition with the TrkC ligands NT-3, 3.5 nM (green) or IY-IY, 20 μM (blue) on cells expressing TrkC. The concentration of NT-3 and IY-IY were kept constant throughout the experiments. Error bars were based on 3 runs.

Figure 4

a Confocal imaging of: first row, the featured targeting ligand on TrkC⁺ cells showing the compound is internalized; second row, the negative control YI-YI-PDT is not localized under the same conditions; third row, the featured agent is not observed in TrkC⁻ cells under the same conditions. b Quantitative indications of the fluorescence intensity in each of the three experiments described above (error bars from 100 cells).

Figure 4

a Confocal imaging of: first row, the featured targeting ligand on TrkC⁺ cells showing the compound is internalized; second row, the negative control YI-YI-PDT is not localized under the same conditions; third row, the featured agent is not observed in TrkC⁻ cells under the same conditions. b Quantitative indications of the fluorescence intensity in each of the three experiments described above (error bars from 100 cells).

Figure 5

IY-IY-PDT colocalizes with LysoTracker Red in TrkC⁺ cells. Throughout, the concentration of the agent used was 1 μM.

Figure 6

Photoinduced cytotoxicity assays. IY-IY-PDT shows high cytotoxicity for the TrkC expressing cells, NIH3T3-TrkC (red) and SY5Y (blue) compared to non-TrkC cells, NIH3T3-WT (purple). The non-targeting ligand, YI-YI-PDT (green) is significantly less photocytotoxic. Error bars were based on 3 runs.

Scheme 1

Synthesis of IY-IY-PDT and YI-YI-PDT