In vitro cellular uptake and dimerization of signal transducer and activator of transcription-3 (STAT3) identify the photosensitizing and imaging-potential of isomeric photosensitizers derived from chlorophyll-a and bacteriochlorophyll-a

Abstract

Among the photosensitizers investigated, both ring-D and ring-B reduced chlorins containing the m-iodobenzyloxyethyl group at position-3 and a carboxylic acid functionality at position-17(2) showed the highest uptake by tumor cells and light-dependent photoreaction that correlated with maximal tumor-imaging [positron emission tomography (PET) and fluorescence] and long-term photodynamic therapy (PDT) efficacy in BALB/c mice bearing Colon26 tumors. However, among the ring-D reduced compounds, the isomer containing the 1'-m-iobenzyloxyethyl group at position-3 was more effective than the corresponding 8-(1'-m-iodobenzyloxyethyl) derivative. All photosensitizers showed maximum uptake by tumor tissue 24 h after injection, and the tumors exposed with light at low fluence and fluence rates (128 J/cm(2), 14 mW/cm(2)) produced significantly enhanced tumor eradication than those exposed at higher fluence and fluence rate (135 J/cm(2), 75 mW/cm(2)). Interestingly, dose-dependent cellular uptake of the compounds and light-dependent STAT3 dimerization have emerged as sensitive rapid indicators for PDT efficacy in vitro and in vivo and could be used as in vitro/in vivo biomarkers for evaluating and optimizing the in vivo treatment parameters of the existing and new PDT candidates.

Figure 1

Various approaches of structural modification of iodinated PS: (X). modification of methyl ester functionality; (Y) effect of the position of (1′-m-iodobenzyloxyethyl) substituent and (Z) effect of ring-B vs. ring-D reduced PSs in biological efficacy.

Figure 2

¹H NMR spectra of ring-D vs. ring-B reduced chlorins 1 and 20 respectively

Figure 3

(A) Electronic absorption spectra of isomers 4, 12 and 23 at equimolar concentration (6.5 μM) in 17% bovine calf serum (BCS). (B) Rate of photobleaching of bacteriochlorins 4, 12 and 23 in 17% BCS. The photosensitizers at equimolar concentration (6.5 μM) were irradiated with light (light dose:75 mW/cm²) and the spectra were taken at variable time points. The normalized long wavelength absorption values were plotted with time showing a comparative rate of photobleaching of the photosensitizers by reactive oxygen species (mainly singlet oxygen) produced in-situ.

Figure 4

PS-specific uptake, photoreaction and post-PDT survival. Replicate 24-well culture plates of BCC1 cells were incubated for 30 min in serum-free DMEM containing 200 nM (A) or for 2 h with serum-free DMEM containing 2-fold serially diluted preparations (B&C) of the PSs indicated at the top. The cells were washed free of PS and imaged at 640X magnification under phase contrast and HPPH fluorescence (A). The plates treated with the serially diluted PSs were illuminated for 9 min with 665 nm light yielding a fluence of 3 J/cm². The cell monolayers in one cultures plate were immediately extracted. Equal aliquots of the extracts were analyzed on western blots for the level of immune detectable STAT3, EGFR and phosphorylated and total p38 MAPK (B). The oxidative crosslinking of STAT3 was determined by densitometric scanning of the bands representing monomeric and homodimeric complex I. The percent conversion of STAT3 to the dimeric form is indicated at the top of panel “B”. The second culture plate was changed to DMEM containing 10% fetal calf serum immediately after light treatment and cultured for an additional 24 h period. The number of surviving, trypan blue-excluding cells were counted in a hemocytometer and expressed relative to the number of cells in the control (C).

Figure 5

Specific patterns of uptake and subcellular distribution of the PSs. A, BCC1 cells were incubated for 30 min in serum-free DMEM containing 3.2 μM PS 1 or 0.2 μM PS 4. The cells were washed and images of the cell monolayers were recorded at 640X magnification under phase contrast or HPPH fluorescence. The cultures were incubated in PS- and serum-free DMEM for an additional 4 h period. The cells were imaged again under identical condition as before. B, BCC1 cells were incubated for 24 h in either serum-free DMEM or DMEM with 10% fetal calf serum, both containing 3.2 μM PS 7. The cells were washed free of PS and imaged a 640X magnification as the cells in A.

Figure 6

(A) In vitro photosensitizing efficacy of PS 1, 4 and 7. (B) PS 4, 12 and 23. Colon26 cells were incubated with 0.3 μM (A) and 0.0625 μM (B) of PSs respectively in growth medium for 24 h. The cells were treated at the peak absorption wavelength with variable light doses and cultured for additional 48h post-treatment. (C) PS uptake of compounds 4, 12 and 23 in Colon26 cells 24 h post incubation was determined by flow cytometry.

Figure 7

(A) Comparative in vivo photosensitizing efficacy of PS 1, PS 4, and PS 7 and (B) PS 4, 12 and 23 in BALB/c mice bearing Colon26 tumors (10 mice/group). The mice were injected with photosensitizers (1.00 μmol/kg) and the tumors were exposed to laser light at 665 nm for compound 1, 4, and 7; 674 nm for compound 23 and 660 nm for compound 12 (light dose: 128J/cm², 14 mW/cm²) at 24h post injection. At day 60, photosensitizers 4 and 23 gave 70% and 60% tumor response respectively (7/10 and 6/10 mice were tumor free), whereas compound 12 showed limited efficacy.

Figure 8

Whole-body PET images of a BALB/c mouse bearing Colon26 tumor with ¹²⁴I- PS 6 (A) taken at 24, 48 and 72 h post-injection. The maximum uptake of the PS was observed at 24h postinjection. However, the best contrast was obtained at 72h for compound PS 6 postinjection.

Figure 9

Comparative biodistribution of ¹²⁴I following injection of PS 3 (A) and PS 6 (B) (100 μCi) in BALB/c mice bearing Colon 26 tumors (3 mice/time point) at 24, 48 and 72 h post-injection. Replacing the methyl ester functionality in PS 3 with carboxylic acid functionality (PS 6) did not make much difference in uptake of the PS in tumor and other organs, except the spleen, where the uptake was significantly reduced.

Figure 10

Biodistribution of ¹⁸F-FDG in BALB/c mice (3 mice/group) bearing Colon26 tumors at 2 h post-injection. The tumor uptake ratio is also presented in tabulated form.

Figure 11

Whole body fluorescence reflectance images (FRI) of representative BALB/c mice implanted with C-26 tumors on the shoulder with compounds 4, 12 and 23 respectively at variable time points with a therapeutic dose (1 μmol kg⁻¹). (A): 24 h p.i.; (B): 48 h p.i. (C) 72 h p.i. (D) Average Fluorescent Intensity (AFU) of 3 mice+/− SD of a ROI (20 mm diameter) over the tumor in AU, arbitrary units.

Figure 12

Whole body fluorescence reflectance images (FRI) of representative BALB/c mice implanted with Colon26 tumors on the shoulder with compounds 4 and 23 at 24 h post-injection (dose: 1 μmol kg −1). λex = 665 and 674 nm for PS 4 and 23 respectively; λem = 710 nm). (A) PS 4; (B) PS 23; tumor was removed and skin was flapped back to expose the residual tumor. The two PSs 4 and 23 exhibiting the highest in vivo phototoxicity were injected intravenously into mice bearing colon 26 tumors to confirm that the recorded fluorescence coincided with the actual tumor mass. After 24 h, the tumors were surgically removed from the primary tumor site and imaged tumor was imaged (Figure 13). The results clearly indicate that most of the fluorescence was confined to the tumor with only low fluorescence associated with the skin section.

Scheme 1

Modification of methyl ester functionality.

Scheme 2

Synthesis of 8-(1′-m-iodobezyloxyethyl)pyropheophorbide 11 and 12 and the 8-Vinyl analog 16.

Scheme 3

Synthetic approaches for the preparation of ring-B reduced 3-(1′-m-iodobenzyloxyethyl) chlorins from bacteriopyropheophorbide-a.