Phenolo-Cyanine Dyes for Fluorescent Monitoring of Targeted Drug Delivery

Drug delivery platforms comprising a switchable fluorescent reporter, anti-cancer drug and targeting carrier (peptide or antibody) are used for targeted drug delivery (TDD) including monitoring of drug release. We synthesized novel phenolo-cyanine dyes of general formula 1 and evaluated them as switchable reporters for TDD monitoring. Acetyl group at the phenolic oxygen was utilized to simulate a cleavable drug moiety; and sulfo groups were used to increase hydrophilicity of the dyes. Carboxylic functionalities can be used for binding of these drug—dye conjugates to targeting carriers. Principle of functioning of switchable dyes consists in the noticeable increase in their fluorescence intensity upon the drug group cleavage, which is due to transformation of the phenolic moiety (dye 1) into the quinone structure (dye 2).

We found that due to an unexpected spirocyclization, dyes 1 can form not only the anticipated fluorescent dye 2 but also non-fluorescent spiropyran 3, which hampers the fluorescence-based monitoring. The electron withdrawing substituents R¹ facilitate undesirable spirocyclization while the electron donative groups slow down this process.

In the example of sulfo group and methylencarboxylic group (R¹) introduced in the position 5 of both indolenine moieties we demonstrated that the electron withdrawing substituents decrease stability of phenolic ester bond towards hydrolytic cleavage while the electron donative substituents stabilize this bond. As compared to the dye 1 with R¹ = H, the cleavage rate increases by factor of 3.5 for 5‑sulfonated derivative (R¹ = SO₃H) and decreases by factor of 3.6 for 5‑methylencarboxylic derivative (R¹ = -CH₂COOH). 5-methylencarboxylic substituent increases the dynamic range of changing the fluorescence intensity by factor of 14, which is extremely important for TDD monitoring.

To conclude, we developed novel, long-wavelength switchable reporter — phenolo-cyanine dye containing 5‑methylencarboxylic and N‑propylsulfonic groups — with increased brightness and dynamic range, which has high potential for fluorescence-based TDD monitoring.