Effect of Metalation on Porphyrin-Based Bifunctional Agents in Tumor Imaging and Photodynamic Therapy

Abstract

Herein we report the syntheses and comparative photophysical, electrochemical, in vitro, and in vivo biological efficacy of 3-(1'-hexyloxy)ethyl-3-devinylpyropheophorbide-cyanine dye (HPPH-CD) and the corresponding indium (In), gallium (Ga), and palladium (Pd) conjugates. The insertion of a heavy metal in the HPPH moiety makes a significant difference in FRET (Förster resonance energy transfer) and electrochemical properties, which correlates with singlet oxygen production [a key cytotoxic agent for photodynamic therapy (PDT)] and long-term in vivo PDT efficacy. Among the metalated analogs, the In(III) HPPH-CD showed the best cancer imaging and PDT efficacy. Interestingly, in contrast to free base HPPH-CD, which requires a significantly higher therapeutic dose (2.5 μmol/kg) than imaging dose (0.3 μmol/kg), the corresponding In(III) HPPH-CD showed excellent imaging and therapeutic potential at a remarkably low dose (0.3 μmol/kg) in BALB/c mice bearing Colon26 tumors. A comparative study of metalated and corresponding nonmetalated conjugates further confirmed that STAT-3 dimerization can be used as a biomarker for determining the level of photoreaction and tumor response.

Figure 1.

Electronic absorption spectrum of HPPH-CD conjugate in methanol. The part of the spectrum presented in red corresponds to the HPPH moiety, whereas the green band belongs to the CD part of the conjugate.

Figure 2.

In vitro photobleaching of compounds 4 (free-base, red), 5 (In, green), and 7 (Pd, blue) in methanol. Absorbance at equimolar concentration (5 μM) in methanol of each compound was measured prior to (solid lines) and after 10 min (dotted lines) of irradiation with 532 nm laser light (125 mW).

Figure 3.

A. Conversion of 4 and 5 to 8a and 8b. B. Kinetic plots of cyanine and porphyrin absorbance (825 and 655 nm, respectively) upon exposing 20 μM solutions (3:1 MeOH:H₂O) of 4 and 5 to 15 mW/cm² light (690 or 780 nm) for the indicated times. C. Relative spectral ion counts of identical solutions of 4, 5, 8a, and 8b over the same time course. Ion counts were determined at each time point relative to an internal standard [3-(3,3-dimethyl-2-oxoindolin-1-yl)propane-1-sulfonate]. The observed ion for 5 is 1646 (M–Cl)+, which corresponds to loss of chloride.

Figure 4.

FRET analysis of PS and CD moieties as a nonmetalated analog 4 (red), indium complex 5 (green), and palladium complex 7 (blue). CD alone (2) is shown in black to show baseline CD fluorescence when excited with 532 nm light. All compounds were dissolved in methanol (5 μM), excited at 532 nm, and fluorescence emission past 800 nm collected in 2 nm steps.

Figure 5.

A. Transient absorption spectra of 4 in deaerated DMSO after femtosecond laser excitation at 410 nm. B,C. Time profiles at 586 nm from 0 to 100 ps and from 0 to 3000 ps.

Figure 6.

A. Transient absorption spectra of 5 in deaerated DMSO after femtosecond laser excitation at 410 nm. B, C. Time profiles at 586 nm from 0 to 100 ps and from 0 to 3000 ps.

Figure 7.

Cyclic voltammograms of investigated compounds in DMSO.

Figure 8.

Thin-layer UV–vis spectral changes at controlled potential reduction of compounds 4 and 5 in DMSO.

Figure 9.

In vitro PDT activity of compounds free-base 4 (red), indium complex 5 (green), gallium complex 6 (orange), and palladium 7 (blue) via the cell viability MTT assay in colon 26 cells. Cells were incubated with each compound in 0.3 μM concentration for 24 h and then irradiated with the appropriate wavelength of light (4 = 665 nm, 5 = 651 nm, 6 = 654 nm, 7 = 640 nm) at various doses of light from 0 to 3.0 J/cm². Values are expressed as cell percent survival of the vehicle-treated controls (set to 100%). Data represents the average of at least 3 experiments; error bars represent standard deviation.

Figure 10.

A. Identification of PS-directed photoreaction by STAT3 cross-linking. BCC1 cells were incubated for 2.5 h with culture medium containing 10% fetal bovine serum and 400 nM of the indicated compounds. The cells were washed and then exposed to light at their compound’s PS respective λ_max yielding at a fluence of 3 J/cm². Immediately after light treatment, the cells were extracted and proteins analyzed by immunoblotting for STAT3. The conversion of monomeric STAT3 to cross-linked dimer complex I was determined and expressed as a percentage of the total STAT3 (as indicated at the top of each lane). B. Subcellular localization of HPPH, In-HPPH, compounds 4 (HPPH-CD)and 5 (In-HPPH-CD). After incubation for 4.5 h, the cells were imaged under an inverted fluorescent phase microscope at 400.

Figure 11.

Whole body fluorescence optical imaging of free-base 4, In complex 5, Ga complex 6, and Pd complex 7 at 24 h post-injection under similar parameters. BALB/c mice (3 per group) bearing Colon26 tumors were injected intravenously with 0.3 μmol/kg of each compound, with one mouse serving as an uninjected control. Images were acquired with a Nuance camera at 782 nm excitation and >830 nm emission using 800/830 nm long-pass filters. Fluorescence intensity scaling is identical in all images. M = BALB/c mice bearing Colon26 tumors.

Figure 12.

In vivo photobleaching of the CD moiety after excitation of In complex 5 (green line), Ga complex 6 (orange), and Pd complex 7 (blue) at a λ_max of 651, 654, and 640 nm, respectively.Three BALB/c mice per group bearing Colon26 tumors were irradiated 24 h post-injection (0.3 μmol/kg solution) at 135 J/cm². Mice were imaged at regular intervals over a 30 min period and the fluorescence intensity of the CD moiety plotted relative to the preirradiated signal. The results are from a single experiment. Means and standard deviations represent average data from the 3 animals.

Figure 13.

In vivo PDT evaluation of metalated analogs: vehicle alone (black, n = 5), indium complex 5 (green, n = 10, 0.3 μmol/kg), gallium complex 6 (orange, n = 3, 1.0 μmol/kg), and palladium complex 7 (blue, n = 3, 0.3 μmol/kg). BALB/c mice with Colon26 tumors were treated with light at an appropriate wavelength (vehicle control = 651 nm, 5 = 651 nm, 6 = 654 nm, 7 = 640 nm) at 135 J/cm²/ 75 mW/C m² (light fluence and fluence rate) at 24 h post-injection.

Scheme 1.

General Procedure for the Synthesis of Metalloporphyrin–Cyanine Dye Conjugates