Effect of chirality on cellular uptake, imaging and photodynamic therapy of photosensitizers derived from chlorophyll-a

Abstract

We have previously shown that the (124)I-analog of methyl 3-(1'-m-iodobenzyloxy) ethyl-3-devinyl-pyropheophorbide-a derived as racemic mixture from chlorophyll-a can be used for PET (positron emission tomography)-imaging in animal tumor models. On the other hand, as a non-radioactive analog, it showed excellent fluorescence and photodynamic therapy (PDT) efficacy. Thus, a single agent in a mixture of radioactive ((124)I-) and non-radioactive ((127)I) material can be used for both dual-imaging and PDT of cancer. Before advancing to Phase I human clinical trials, we evaluated the activity of the individual isomers as well as the impact of a chiral center at position-3(1) in directing in vitro/in vivo cellular uptake, intracellular localization, epithelial tumor cell-specific retention, fluorescence/PET imaging, and photosensitizing ability. The results indicate that both isomers (racemates), either as methyl ester or carboxylic acid, were equally effective. However, the methyl ester analogs, due to subcellular deposition into vesicular structures, were preferentially retained. All derivatives containing carboxylic acid at the position-17(2) were noted to be substrate for the ABCG2 (a member of the ATP binding cassette transporters) protein explaining their low retention in lung tumor cells expressing this transporter. The compounds in which the chirality at position-3 has been substituted by a non-chiral functionality showed reduced cellular uptake, retention and lower PDT efficacy in mice bearing murine Colon26 tumors.

Keywords: Cell-specificity; Chlorophyll-a; Imaging; Photodynamic therapy; Photosensitizer.

Figure 1

HPLC was carried out using a Waters Delta 600 system consisting of the 600 Controller, 600 Fluid Handling Unit and 996 Photodiode Array Detector equipped with a Chiralpak IB column with dimensions 4.6 × 250 mm, 5 µm particle size. The mobile phase was isocratic: 40% ethyl acetate–60% hexane at a flow rate of 1.0 ml/min. Chromatographs indicate: A, compound 2; B, compound 3; C, compound 4; and D, compound 9. The retention times for isomers 3 and 4 were 5.9 and 6.6 min, respectively. The retention time for nonchiral compound 9 was 8.0 min.

Figure 2

Partial ¹H NMR spectra show slight differences in the chemical shifts of the 3¹ methyl proton doublets of compound 3 (B) and compound 4 (C). In compound 2 (A) both doublets are present, as expected for the mixture containing both forms. In both (B) and (C), the presence of only one set of peaks suggests that 3 and 4 are isomerically pure.

Figure 3

Uptake and intracellular localization of the compounds 2–6 in colon 26 cells after 24 hour incubation in medium containing 0.8 µM compounds. (A): Total intracellular uptake determined by fluorescence. (B): Representative images from flow cytometry analysis (Image Stream) comparing subcellular distribution of compounds and organelle-tracking dyes. (C): Similarity Score as a measure for co-localization of the compounds with the lysosomal and mitochondrial markers (the higher positive the score the more closely the fluorescent probes co-localize). Data represent the means ±SD of replicate samples from 3 separate experiments).

Figure 4

Binding and intracellular retention of compounds 3–6 (PS 3–6) by human lung tumor fibroblasts. Subconfluent cultures of fibroblast were incubated in medium containing 3.2 µM of the indicated compunds. Binding to the cells was determined by 30-min exposure at 0°, uptake by incubation of the cells for 4 hours at 37° and retention by culturing in compound-free medium for 20 hours. Cell-associatied fluorescence of the compounds was visualized by microscopy (400× magnification).

Figure 5

Subcellular localization of compounds 2 and 2a in lung tumor fibroblasts. Primary cultures of tumor-derived fibroblasts were transduced with vectors encoding ER-GFP, Golgi-GFP or Peroxisome-RFP. After 24 h, the cells were incubated for 4 hours with compound 2 or compound 2a. Thirty min prior to the end of incubation, separate cultures were stained with Mito-tracker green or Lysotracker green. All cultures were analyzed by phase contrast and fluorescent microcopy at 400×. All organelle markers are colorized in green, and fluorescence for compounds 2 and 2a is shown in red.

Figure 6

Lung tumor cell-specific retention of compounds 2 and 2a (PS 2 and PS 2a). Co-cultures of primary lung squamous carcinoma cells and CSFE-labeled stromal fibroblasts were incubated for 4 h with culture medium containing 3.2 µM PS 2 or PS 2a followed by 6 and 24 h chase in PS-free medium. The culture-associated fluorescence was recorded at the times indicated by microscopy.

Figure 7

Cellular export of carboxylic acid derivatives controled by ABCG2. Primary cultures of ABCG2-positive L352 epithelial cells were incubated for 30 min at 37° in serum-free medium containing 3.2 µM compounds 2 (PS 2), 2a (PS 2a) or 9a (PS 9a). The cells were washed and incubated for 4 hours in compound-free medium containing 10% FBS and either 0 or 10 µM imatinib mesylate. Cell-associated compounds were determined by fluorescence microscopy at 400× magnification using identical camera setting.

Figure 8

Effect of the 3’-chiral center on cellular retention in lung tumor cells. Replicate 6-well culture plates of lung tumor epithelial cells were incubated for 4 h in culture medium containing 10% FBS and 800 nM of PS 2, 2a or non-chiral PS (9 and 9a). The cells were washed free of PS and imaged at 400× magnification under fluorescence. Cells were incubated in PS-free medium containing 10% FBS for additional 4 and 24 hours.

Figure 9

Correlation of compound uptake and photoreaction. Confluent epithelial cells were incubated for 4 hours with 800 nM (for fluorescent imaging in A) or 100 nM (for biochemical analysis in B–D) compounds listed at the top. After a 4-h chase period, the cell-associated fluorescence was determined by imaging. The cells in B–D were treated with 665nm light (3 J/cm²), extracted and the relative amount of oxidative crosslinking of STAT3 and level of EGFR and phospho- and total p38 MAPK were determined by immunoblotting.

Figure 10

In vivo fluorescence detection of compound PS 2 and its isomers 3 and 4 accumulation in the colon26 tumors. Representative Images of 1 µmole/kg PS 2 injected i.v. and imaged at 24, 48 and 72 hours post injection (excitation wavelength - 675 nm, emission wavelength −720 nm). B: Data represent the mean fluorescence expressed as total radiant efficiency ±SD of 3 mice/group.

Figure 11

Comparative in vivo photosensitizing efficacy of PSs in BALB/c mice bearing Colon 26 tumors at variable light doses. In four separate sets of experiments, tumor-bearing mice were subjected to following treatments: all PS injections were i.v. at 1.00 µmol/kg with light treatment of the tumor 24 h later. (A): PS 2 (with laser light at 665 nm using two conditions: 135 J/cm², 75mW/cm² () and 128J/cm², 14 mW/cm², ()]. (B): PS 2 and the corresponding R- and S- isomers 3, 4; (C): isomers 5 and 6 and, (D): PS 2 and PS. 9 (achiral at position-3). All animals in B to D were treated with 665 nm light (128J/cm², 14 mW/cm²), Tumor growth was monitored daily for 60 days.

Figure 12

PDT efficacy of Compound 2 (PS2) and Photofrin (PFII) on human NSCLC xenograft tissue. Xenograft tissue was dissociated, plated onto collagen-1 matrix and cultured for 4 days. The tumor cells form clusters segregated from stromal cells. The cultures were treated for 4 hours with medium containing 6µg/ml PFII or 1µg/ml PS 2. The cell-associated PS fluorescence was recorded immediately after uptake and after a 24-hour chase period. The phase microscopic and corresponding fluorescent images at 100× are reproduced.

Figure 13

Coronal view PET images of a BALB/c mouse bearing Colon26 tumor on the right shoulder with labeled ¹²⁴I-PS 12 or 13 (50µCi). The images were acquired for 30 min at 24, 48, and 72 hours post-injection. The tumor was identified to be within the region defined by a cylinder indicated by the blue rectangle in each image. The color palette (shown on the right) for each image shown was scaled to the min/max of each data set. The tumor uptake (3 mice/group) of the isomers at various time points (24, 48 and 72 hours) is represented in bar graph and shows similar pattern of uptake and clearance. The absolute tumor-uptake was higher at 24 hours post-injection, but tumor contrast was better at 48 and 72 hours post-injection, which could be due to faster clearance of the compounds from other organs than tumor.

Scheme 1

Synthesis of iodinated photosensitizers with and without the presence of a chiral center at position-3.