Synthesis of Tumor-avid Photosensitizer-Gd(III)DTPA conjugates: impact of the number of gadolinium units in T1/T2 relaxivity, intracellular localization, and photosensitizing efficacy

Abstract

To develop novel bifunctional agents for tumor imaging (MR) and photodynamic therapy (PDT), certain tumor-avid photosensitizers derived from chlorophyll-a were conjugated with variable number of Gd(III)aminobenzyl DTPA moieties. All the conjugates containing three or six gadolinium units showed significant T(1) and T(2) relaxivities. However, as a bifunctional agent, the 3-(1'-hexyloxyethyl)pyropheophorbide-a (HPPH) containing 3Gd(III) aminophenyl DTPA was most promising with possible applications in tumor-imaging and PDT. Compared to HPPH, the corresponding 3- and 6Gd(III)aminobenzyl DTPA conjugates exhibited similar electronic absorption characteristics with a slightly decreased intensity of the absorption band at 660 nm. However, compared to HPPH, the excitation of the broad "Soret" band (near 400 nm) of the corresponding 3Gd(III)aminobenzyl-DTPA analogues showed a significant decrease in the fluorescence intensity at 667 nm.

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Figure 1

Comparative absorption (A) and fluorescence spectra (B) of HPPH and the corresponding 3 Gd(III)aminobenzyl DTPA 3 and 6Gd(III)aminobenzyl DTPA conjugate 15 in methanol (conc. 19.6 μM).

Figure 2

Comparative photobleaching characteristics of HPPH and the corresponding 3 and 6Gd(III) aminobenzyl DTPA 3 and 15 respectively at (9.8 M) in 17% BCS. The solutions were exposed with a laser light (500 mW, 665 nm, 75 mW/cm²) and the absorption peak at 665 nm were recorded at variable time points.

Figure 3

In vitro photosensitizing activity of photosensitizer-Gd(III)ADTPA conjugates (4.0 μM) in RIF cells. The cells were incubated for 24h and then exposed to light at a dose of 3.2 mW/cm². Control cells were exposed to light only.

Figure 4

False-color images showing subcellular localization of HPPH and compound 3 (red) in Colon26 cells after 4h (B) or 24 h incubations (A and C) respectively. Localization was determined by fluorescence microscopy by co-incubation with fluorescent organelle probes (green). Subcellular areas of co-localization in the overlay of compound 3 or HPPH with the probes for are yellowish orange. A, HPPH; B and C, compound 3; D, MitoTracker Green; E and F, Bodipy ceramide C5-albumin complex; G, overlay of HPPH and MitoTracker Green; H and I overlays of compound 3 and Bodipy ceramide C5-albumin complex