Limits...
Syntheses and Photodynamic Activity of Pegylated Cationic Zn(II)-Phthalocyanines in HEp2 Cells.

Ongarora BG, Hu X, Verberne-Sutton SD, Garno JC, Vicente MG - Theranostics (2012)

Bottom Line: The most phototoxic compounds were found to be the α-substituted Pcs.The β-substituted ZcPcs 6b and 4b accumulated the most within HEp2 cells but had low photocytoxicity (IC(50) > 100 μM at 1.5 J/cm(2)), possibly as a result of their lower electron density of the ring and more extended conformations compared with the α-substituted Pcs.The results show that the charge distribution about the Pc macrocycle and the intracellular localization of the cationic ZnPcs mainly determine their photodynamic activity.

View Article: PubMed Central - PubMed

Affiliation: Louisiana State University, Department of Chemistry, Baton Rouge LA, 70803, USA.

ABSTRACT
Di-cationic Zn(II)-phthalocyanines (ZnPcs) are promising photosensitizers for the photodynamic therapy (PDT) of cancers and for photoinactivation of viruses and bacteria. Pegylation of photosensitizers in general enhances their water-solubility and tumor cell accumulation. A series of pegylated di-cationic ZnPcs were synthesized from conjugation of a low molecular weight PEG group to a pre-formed Pc macrocycle, or by mixed condensation involving a pegylated phthalonitrile. All pegylated ZnPcs were highly soluble in polar organic solvents but were insoluble in water; they have intense Q absorptions centered at 680 nm and fluorescence quantum yields of ca. 0.2 in DMF. The non-pegylated di-cationic ZnPc 6a formed large aggregates, which were visualized by atomic force microscopy. The cytotoxicity, cellular uptake and subcellular distribution of all cationic ZnPcs were investigated in human carcinoma HEp2 cells. The most phototoxic compounds were found to be the α-substituted Pcs. Among these, Pcs 4a and 16a were the most effective (IC(50) ca. 10 μM at 1.5 J/cm(2)), in part due to the presence of a PEG group and the two positive charges in close proximity (separated by an ethylene group) in these macrocycles. The β-substituted ZcPcs 6b and 4b accumulated the most within HEp2 cells but had low photocytoxicity (IC(50) > 100 μM at 1.5 J/cm(2)), possibly as a result of their lower electron density of the ring and more extended conformations compared with the α-substituted Pcs. The results show that the charge distribution about the Pc macrocycle and the intracellular localization of the cationic ZnPcs mainly determine their photodynamic activity.

No MeSH data available.


Related in: MedlinePlus

Subcellular localization of ZnPc 16a in HEp2 cells at 10 μM for 6 h. (a) Phase contrast, (b) Overlay of 16a fluorescence and phase contrast, (c) ER Tracker Blue/White fluorescence, (e) MitoTracker green fluorescence, (g) BODIPY Ceramide fluorescence, (i) LysoSensor green fluorescence, and (d, f, h, j) overlays of organelle tracers with 16a fluorescence. Scale bar: 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3475216&req=5

Figure 6: Subcellular localization of ZnPc 16a in HEp2 cells at 10 μM for 6 h. (a) Phase contrast, (b) Overlay of 16a fluorescence and phase contrast, (c) ER Tracker Blue/White fluorescence, (e) MitoTracker green fluorescence, (g) BODIPY Ceramide fluorescence, (i) LysoSensor green fluorescence, and (d, f, h, j) overlays of organelle tracers with 16a fluorescence. Scale bar: 10 μm.

Mentions: The biological properties of the cationic ZnPcs including time-dependent cellular uptake, cytotoxicity and intracellular localization, were investigated in human carcinoma HEp2 cells. The cytotoxicity was evaluated using Promega's Cell Titer Blue viability assay, as we have previously reported 28,35,36,39,44 at concentrations up to 400 μM for each di-cationic ZnPc and for the pegylated, neutral ZnPc 2b (see Supplementary Material: Figure S9). The time-dependent uptake into HEp2 cells was investigated at a concentration of 10 μM of each Pc up to 24 h (Figure 3). The subcellular sites of localization were observed by fluorescence microscopy, 6 h after exposure of HEp2 cells to each ZnPc (see Figures 4-6 and Supporting Information, Supplementary Material: Figures S1-S8). Co-localization experiments were conducted using the organelle specific fluorescent tracers: ER Tracker Blue/White (endoplasmic reticulum, ER), MitoTracker Green (mitochondria), BODIPY Ceramide (Golgi) and LysoSensor Green (lysosomes). Table 2 summarizes the results obtained from these studies. Our results show that all the α-substituted ZnPcs are much more toxic than the corresponding β-substituted ZnPcs, both in the dark and upon exposure to approx. 1.5 J/cm2 light dose. We had previously observed that the cytotoxicity of cationic Pcs depends on the substitution at the macrocycle periphery, and that the α-substituted compounds tend to be more toxic than the corresponding β-substituted Pcs 35. This might be due to their increased electron density of the ring and distinct conformations, as shown in Supplementary Material: Figure S21; the β-substituted di-cationic ZnPcs tend to adopt more extended conformations than the α-substituted analogues. All ZnPcs had very low toxicity in the dark (IC50 > 180 μM), in particular the branched dicationic Pcs 9a,b and 11a,b which showed remarkable low dark toxicity (IC50 > 400 μM), maybe due to their very low uptake into cells (see Figure 3). Upon irradiation with low light dose (~1.5 J/cm2) all β-substituted ZnPcs were non-toxic up to 100 μM concentrations, with the exception of 11b, which was moderately phototoxic (IC50 = 47 μM). The most phototoxic compounds were the α-substituted ZnPcs 4a, 6a, 9a, 11a and 16a, with determined IC50 values (calculated from dose-response curves, see Supporting Information) of 10.7, 14.8, 28.8, 12.7 and 8.7 μM, respectively. Of this series, Pcs 4a, 11a and 16a are the most promising for PDT applications due to their high ratio (>25) of dark/photo cytotoxicity and high phototoxicity. It is interesting to note that the most phototoxic Pcs (4a and 16a) contain a PEG group, the two positive charges in close proximity (separated only by an ethylene group), and both localize in the cell ER. It is possible that pegylation of Pc macrocycles favors intracellular localization in the ER, as we have previously observed 38, whereas the positive charges might favor localization at the plasma membrane and subcellularly in mitochondria and lysosomes 44-47. On the other hand, the β-substituted ZcPcs 6b and 4b accumulated within HEp2 cells to a much higher extent than all other Pcs. The cellular uptake of the non-pegylated Pc 6b was the fastest of all ZnPcs, although it slowed down to a plateau 8 h after exposure, while Pc 4b continued to accumulate within cells up to the 24 h period investigated, probably as a result of its PEG group. Furthermore, the di-cationic Pcs bearing the two charges in close proximity on the same chain, showed increased uptake compared with the neutral Pc-PEG 2b, as well as to the cationic Pcs 9a,b and 11a,b bearing the charges on two side branches. All cationic ZnPcs localized in multiple sites within the cell, with exception of 9b which was mainly found in lysosomes (Table 2). In addition, ZnPcs 4a, 6a, 11a and 16b were observed at the plasma membrane. The most phototoxic compounds 4a, 11a and 16a all localized within the ER, an important organelle that regulates protein synthesis and stress responses, potentially leading to PDT-induced cell apoptosis 48,49. Our results show that the intracellular localization of the ZnPcs, rather than the extent of their cellular uptake, and their charge distribution mainly determine their photodynamic activity, probably as a result of their different interactions with intracellular components.


Syntheses and Photodynamic Activity of Pegylated Cationic Zn(II)-Phthalocyanines in HEp2 Cells.

Ongarora BG, Hu X, Verberne-Sutton SD, Garno JC, Vicente MG - Theranostics (2012)

Subcellular localization of ZnPc 16a in HEp2 cells at 10 μM for 6 h. (a) Phase contrast, (b) Overlay of 16a fluorescence and phase contrast, (c) ER Tracker Blue/White fluorescence, (e) MitoTracker green fluorescence, (g) BODIPY Ceramide fluorescence, (i) LysoSensor green fluorescence, and (d, f, h, j) overlays of organelle tracers with 16a fluorescence. Scale bar: 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475216&req=5

Figure 6: Subcellular localization of ZnPc 16a in HEp2 cells at 10 μM for 6 h. (a) Phase contrast, (b) Overlay of 16a fluorescence and phase contrast, (c) ER Tracker Blue/White fluorescence, (e) MitoTracker green fluorescence, (g) BODIPY Ceramide fluorescence, (i) LysoSensor green fluorescence, and (d, f, h, j) overlays of organelle tracers with 16a fluorescence. Scale bar: 10 μm.
Mentions: The biological properties of the cationic ZnPcs including time-dependent cellular uptake, cytotoxicity and intracellular localization, were investigated in human carcinoma HEp2 cells. The cytotoxicity was evaluated using Promega's Cell Titer Blue viability assay, as we have previously reported 28,35,36,39,44 at concentrations up to 400 μM for each di-cationic ZnPc and for the pegylated, neutral ZnPc 2b (see Supplementary Material: Figure S9). The time-dependent uptake into HEp2 cells was investigated at a concentration of 10 μM of each Pc up to 24 h (Figure 3). The subcellular sites of localization were observed by fluorescence microscopy, 6 h after exposure of HEp2 cells to each ZnPc (see Figures 4-6 and Supporting Information, Supplementary Material: Figures S1-S8). Co-localization experiments were conducted using the organelle specific fluorescent tracers: ER Tracker Blue/White (endoplasmic reticulum, ER), MitoTracker Green (mitochondria), BODIPY Ceramide (Golgi) and LysoSensor Green (lysosomes). Table 2 summarizes the results obtained from these studies. Our results show that all the α-substituted ZnPcs are much more toxic than the corresponding β-substituted ZnPcs, both in the dark and upon exposure to approx. 1.5 J/cm2 light dose. We had previously observed that the cytotoxicity of cationic Pcs depends on the substitution at the macrocycle periphery, and that the α-substituted compounds tend to be more toxic than the corresponding β-substituted Pcs 35. This might be due to their increased electron density of the ring and distinct conformations, as shown in Supplementary Material: Figure S21; the β-substituted di-cationic ZnPcs tend to adopt more extended conformations than the α-substituted analogues. All ZnPcs had very low toxicity in the dark (IC50 > 180 μM), in particular the branched dicationic Pcs 9a,b and 11a,b which showed remarkable low dark toxicity (IC50 > 400 μM), maybe due to their very low uptake into cells (see Figure 3). Upon irradiation with low light dose (~1.5 J/cm2) all β-substituted ZnPcs were non-toxic up to 100 μM concentrations, with the exception of 11b, which was moderately phototoxic (IC50 = 47 μM). The most phototoxic compounds were the α-substituted ZnPcs 4a, 6a, 9a, 11a and 16a, with determined IC50 values (calculated from dose-response curves, see Supporting Information) of 10.7, 14.8, 28.8, 12.7 and 8.7 μM, respectively. Of this series, Pcs 4a, 11a and 16a are the most promising for PDT applications due to their high ratio (>25) of dark/photo cytotoxicity and high phototoxicity. It is interesting to note that the most phototoxic Pcs (4a and 16a) contain a PEG group, the two positive charges in close proximity (separated only by an ethylene group), and both localize in the cell ER. It is possible that pegylation of Pc macrocycles favors intracellular localization in the ER, as we have previously observed 38, whereas the positive charges might favor localization at the plasma membrane and subcellularly in mitochondria and lysosomes 44-47. On the other hand, the β-substituted ZcPcs 6b and 4b accumulated within HEp2 cells to a much higher extent than all other Pcs. The cellular uptake of the non-pegylated Pc 6b was the fastest of all ZnPcs, although it slowed down to a plateau 8 h after exposure, while Pc 4b continued to accumulate within cells up to the 24 h period investigated, probably as a result of its PEG group. Furthermore, the di-cationic Pcs bearing the two charges in close proximity on the same chain, showed increased uptake compared with the neutral Pc-PEG 2b, as well as to the cationic Pcs 9a,b and 11a,b bearing the charges on two side branches. All cationic ZnPcs localized in multiple sites within the cell, with exception of 9b which was mainly found in lysosomes (Table 2). In addition, ZnPcs 4a, 6a, 11a and 16b were observed at the plasma membrane. The most phototoxic compounds 4a, 11a and 16a all localized within the ER, an important organelle that regulates protein synthesis and stress responses, potentially leading to PDT-induced cell apoptosis 48,49. Our results show that the intracellular localization of the ZnPcs, rather than the extent of their cellular uptake, and their charge distribution mainly determine their photodynamic activity, probably as a result of their different interactions with intracellular components.

Bottom Line: The most phototoxic compounds were found to be the α-substituted Pcs.The β-substituted ZcPcs 6b and 4b accumulated the most within HEp2 cells but had low photocytoxicity (IC(50) > 100 μM at 1.5 J/cm(2)), possibly as a result of their lower electron density of the ring and more extended conformations compared with the α-substituted Pcs.The results show that the charge distribution about the Pc macrocycle and the intracellular localization of the cationic ZnPcs mainly determine their photodynamic activity.

View Article: PubMed Central - PubMed

Affiliation: Louisiana State University, Department of Chemistry, Baton Rouge LA, 70803, USA.

ABSTRACT
Di-cationic Zn(II)-phthalocyanines (ZnPcs) are promising photosensitizers for the photodynamic therapy (PDT) of cancers and for photoinactivation of viruses and bacteria. Pegylation of photosensitizers in general enhances their water-solubility and tumor cell accumulation. A series of pegylated di-cationic ZnPcs were synthesized from conjugation of a low molecular weight PEG group to a pre-formed Pc macrocycle, or by mixed condensation involving a pegylated phthalonitrile. All pegylated ZnPcs were highly soluble in polar organic solvents but were insoluble in water; they have intense Q absorptions centered at 680 nm and fluorescence quantum yields of ca. 0.2 in DMF. The non-pegylated di-cationic ZnPc 6a formed large aggregates, which were visualized by atomic force microscopy. The cytotoxicity, cellular uptake and subcellular distribution of all cationic ZnPcs were investigated in human carcinoma HEp2 cells. The most phototoxic compounds were found to be the α-substituted Pcs. Among these, Pcs 4a and 16a were the most effective (IC(50) ca. 10 μM at 1.5 J/cm(2)), in part due to the presence of a PEG group and the two positive charges in close proximity (separated by an ethylene group) in these macrocycles. The β-substituted ZcPcs 6b and 4b accumulated the most within HEp2 cells but had low photocytoxicity (IC(50) > 100 μM at 1.5 J/cm(2)), possibly as a result of their lower electron density of the ring and more extended conformations compared with the α-substituted Pcs. The results show that the charge distribution about the Pc macrocycle and the intracellular localization of the cationic ZnPcs mainly determine their photodynamic activity.

No MeSH data available.


Related in: MedlinePlus