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Upconversion nanoparticle-mediated photodynamic therapy induces THP-1 macrophage apoptosis via ROS bursts and activation of the mitochondrial caspase pathway.

Zhu X, Wang H, Zheng L, Zhong Z, Li X, Zhao J, Kou J, Jiang Y, Zheng X, Liu Z, Li H, Cao W, Tian Y, Wang Y, Yang L - Int J Nanomedicine (2015)

Bottom Line: Our results indicated that the levels of ROS were significantly increased in the PDT group, resulting in both MPTP opening and MMP depolarization, which led to apoptosis.The proapoptotic factor Bax subsequently translocates from the cytosol to the mitochondria, resulting in the MPTP opening and cytochrome C release.This study demonstrated the great potential of UCNPs-Ce6-mediated PDT in the treatment of AS.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology, Harbin Medical University, Harbin, People's Republic of China.

ABSTRACT
Atherosclerosis (AS) is the most vital cardiovascular disease, which poses a great threat to human health. Macrophages play an important role in the progression of AS. Photodynamic therapy (PDT) has emerged as a useful therapeutic modality not only in the treatment of cancer but also in the treatment of AS. The purpose of this study was to determine the molecular mechanisms underlying the activity of PDT, using mesoporous-silica-coated upconversion fluorescent nanoparticles encapsulating chlorin e6 (UCNPs-Ce6) in the induction of apoptosis in THP-1 macrophages. Here, we investigated the ability of UCNPs-Ce6-mediated PDT to induce THP-1 macrophage apoptosis by facilitating the induction of reactive oxygen species (ROS) and regulation of mitochondrial permeability transition pore (MPTP) to depolarize mitochondrial membrane potential (MMP). Both Bax translocation and the release of cytochrome C were examined using immunofluorescence and Western blotting. Our results indicated that the levels of ROS were significantly increased in the PDT group, resulting in both MPTP opening and MMP depolarization, which led to apoptosis. In addition, immunofluorescence and Western blotting revealed that PDT induced both Bax translocation and the release of cytochrome C, as well as upregulation of cleaved caspase-9, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase. Therefore, we demonstrated that UCNPs-Ce6-mediated PDT induces apoptosis in THP-1 macrophages via ROS bursts. The proapoptotic factor Bax subsequently translocates from the cytosol to the mitochondria, resulting in the MPTP opening and cytochrome C release. This study demonstrated the great potential of UCNPs-Ce6-mediated PDT in the treatment of AS.

No MeSH data available.


Related in: MedlinePlus

Mesoporous-silica-coated UCNPs as an agent of PDT.Notes: (A) Schematic of mesoporous-silica-coated UCNPs co-loaded with chlorin e6 (Ce6) photosensitizer for PDT. Following excitation at 980 nm, the UCNPs upconverted NIR light to VIS light of specific wavelengths to excite Ce6, and the activated photosensitizer subsequently generated cytotoxic 1O2 for biomolecule modification. (B) Comparison of 1O2 production as determined by the decay of ABDA fluorescence. ABDA fluorescence decay (measured at a peak intensity of 431 nm) was plotted as a function of exposure time (min) to an NIR laser (the experiments for each group were run in triplicate). (C) A comparison of penetration depth as determined via cell viability following PDT using different thicknesses of pigskin. (D) The intracellular accumulation of UCNPs-Ce6 as detected via laser confocal microscopy (Ce6 red fluorescence). **P<0.01, ***P<0.001 vs control group. Scale bar =0.1 mm.Abbreviations: ABDA, 9, 10-anthracenediylbis (methylene) dimalonic acid; UCNPs, upconversion nanoparticles; PDT, photodynamic therapy; NIR, near-infrared; VIS, visible; 1O2, singlet oxygen; 3O2, triplet oxygen; h, hours.
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f1-ijn-10-3719: Mesoporous-silica-coated UCNPs as an agent of PDT.Notes: (A) Schematic of mesoporous-silica-coated UCNPs co-loaded with chlorin e6 (Ce6) photosensitizer for PDT. Following excitation at 980 nm, the UCNPs upconverted NIR light to VIS light of specific wavelengths to excite Ce6, and the activated photosensitizer subsequently generated cytotoxic 1O2 for biomolecule modification. (B) Comparison of 1O2 production as determined by the decay of ABDA fluorescence. ABDA fluorescence decay (measured at a peak intensity of 431 nm) was plotted as a function of exposure time (min) to an NIR laser (the experiments for each group were run in triplicate). (C) A comparison of penetration depth as determined via cell viability following PDT using different thicknesses of pigskin. (D) The intracellular accumulation of UCNPs-Ce6 as detected via laser confocal microscopy (Ce6 red fluorescence). **P<0.01, ***P<0.001 vs control group. Scale bar =0.1 mm.Abbreviations: ABDA, 9, 10-anthracenediylbis (methylene) dimalonic acid; UCNPs, upconversion nanoparticles; PDT, photodynamic therapy; NIR, near-infrared; VIS, visible; 1O2, singlet oxygen; 3O2, triplet oxygen; h, hours.

Mentions: To prepare the UCNPs-Ce6 for the study, we uniformly coated NaYF4 UCNPs with mesoporous silica, in which Ce6 was encased. The irradiation of these UCNPs with a 980 nm laser resulted in the emission of upconversion visible fluorescence at two primary peaks, namely, green (approximately 540 nm) and red (approximately 660 nm). We chose this formulation of UCNPs for our PDT experiments because its 660 nm emission wavelength matched well with the absorption peak of Ce6 (Figure 1A). To confirm the ability of UCNPs-Ce6 to generate 1O2, we used a chemical method using the dye ABDA as an acceptor of 1O2. In the presence of 1O2, the consumption of ABDA results in fluorescence decay of the dye, thereby providing a means of monitoring 1O2 production from UCNPs-Ce6. We measured the extent of ABDA decay in the water in the different treatment groups (Figure 1B). The PDT group demonstrated the highest rate of decay, and this rate was significantly higher than those of control, UCNPs-Ce6 alone, or laser alone, as these groups demonstrated only marginal ABDA decay. To determine whether such decay was a result of the involvement of 1O2, we ran a parallel study in which we added the 1O2 scavenger NaN3 to the suspension of the PDT group. The presence of NaN3 caused a marked decrease in the rate of ABDA decay, thus confirming the participation of 1O2 in this process. Conventional PDT treatment has previously demonstrated low penetration depth, which is an important drawback not only in the treatment of tumors but also in the treatment of AS. We investigated the penetration depth induced by UCNPs-Ce6-mediated PDT. As shown in Figure 2C, the viability of the THP-1 macrophages treated with PDT (16 μg/mL UCNPs-Ce6 plus 60 seconds of 980 nm laser) declined significantly to 67%. However, the addition of pigskin restored cell viability decrease in a thickness-dependent manner, thereby illustrating that the tissues absorbed the 980 nm laser to a certain extent. The 980 nm laser penetrated the 6 mm pigskin and induced cell death. Wang et al22 confirmed that the efficiency of conventional PDT treatment using a 660 nm laser decreases by 80% if the laser is blocked by tissues with a thickness of 3 mm, whereas decreases of only 5% is noted in the setting of NIR-treated PDT. We also indirectly detected the accumulation of UCNPs-Ce6 in the THP-1 macrophages via the fluorescence of Ce6 (red color), and we observed that the fluorescence reached its highest level at 4 hours following incubation (Figure 1D).


Upconversion nanoparticle-mediated photodynamic therapy induces THP-1 macrophage apoptosis via ROS bursts and activation of the mitochondrial caspase pathway.

Zhu X, Wang H, Zheng L, Zhong Z, Li X, Zhao J, Kou J, Jiang Y, Zheng X, Liu Z, Li H, Cao W, Tian Y, Wang Y, Yang L - Int J Nanomedicine (2015)

Mesoporous-silica-coated UCNPs as an agent of PDT.Notes: (A) Schematic of mesoporous-silica-coated UCNPs co-loaded with chlorin e6 (Ce6) photosensitizer for PDT. Following excitation at 980 nm, the UCNPs upconverted NIR light to VIS light of specific wavelengths to excite Ce6, and the activated photosensitizer subsequently generated cytotoxic 1O2 for biomolecule modification. (B) Comparison of 1O2 production as determined by the decay of ABDA fluorescence. ABDA fluorescence decay (measured at a peak intensity of 431 nm) was plotted as a function of exposure time (min) to an NIR laser (the experiments for each group were run in triplicate). (C) A comparison of penetration depth as determined via cell viability following PDT using different thicknesses of pigskin. (D) The intracellular accumulation of UCNPs-Ce6 as detected via laser confocal microscopy (Ce6 red fluorescence). **P<0.01, ***P<0.001 vs control group. Scale bar =0.1 mm.Abbreviations: ABDA, 9, 10-anthracenediylbis (methylene) dimalonic acid; UCNPs, upconversion nanoparticles; PDT, photodynamic therapy; NIR, near-infrared; VIS, visible; 1O2, singlet oxygen; 3O2, triplet oxygen; h, hours.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4447170&req=5

f1-ijn-10-3719: Mesoporous-silica-coated UCNPs as an agent of PDT.Notes: (A) Schematic of mesoporous-silica-coated UCNPs co-loaded with chlorin e6 (Ce6) photosensitizer for PDT. Following excitation at 980 nm, the UCNPs upconverted NIR light to VIS light of specific wavelengths to excite Ce6, and the activated photosensitizer subsequently generated cytotoxic 1O2 for biomolecule modification. (B) Comparison of 1O2 production as determined by the decay of ABDA fluorescence. ABDA fluorescence decay (measured at a peak intensity of 431 nm) was plotted as a function of exposure time (min) to an NIR laser (the experiments for each group were run in triplicate). (C) A comparison of penetration depth as determined via cell viability following PDT using different thicknesses of pigskin. (D) The intracellular accumulation of UCNPs-Ce6 as detected via laser confocal microscopy (Ce6 red fluorescence). **P<0.01, ***P<0.001 vs control group. Scale bar =0.1 mm.Abbreviations: ABDA, 9, 10-anthracenediylbis (methylene) dimalonic acid; UCNPs, upconversion nanoparticles; PDT, photodynamic therapy; NIR, near-infrared; VIS, visible; 1O2, singlet oxygen; 3O2, triplet oxygen; h, hours.
Mentions: To prepare the UCNPs-Ce6 for the study, we uniformly coated NaYF4 UCNPs with mesoporous silica, in which Ce6 was encased. The irradiation of these UCNPs with a 980 nm laser resulted in the emission of upconversion visible fluorescence at two primary peaks, namely, green (approximately 540 nm) and red (approximately 660 nm). We chose this formulation of UCNPs for our PDT experiments because its 660 nm emission wavelength matched well with the absorption peak of Ce6 (Figure 1A). To confirm the ability of UCNPs-Ce6 to generate 1O2, we used a chemical method using the dye ABDA as an acceptor of 1O2. In the presence of 1O2, the consumption of ABDA results in fluorescence decay of the dye, thereby providing a means of monitoring 1O2 production from UCNPs-Ce6. We measured the extent of ABDA decay in the water in the different treatment groups (Figure 1B). The PDT group demonstrated the highest rate of decay, and this rate was significantly higher than those of control, UCNPs-Ce6 alone, or laser alone, as these groups demonstrated only marginal ABDA decay. To determine whether such decay was a result of the involvement of 1O2, we ran a parallel study in which we added the 1O2 scavenger NaN3 to the suspension of the PDT group. The presence of NaN3 caused a marked decrease in the rate of ABDA decay, thus confirming the participation of 1O2 in this process. Conventional PDT treatment has previously demonstrated low penetration depth, which is an important drawback not only in the treatment of tumors but also in the treatment of AS. We investigated the penetration depth induced by UCNPs-Ce6-mediated PDT. As shown in Figure 2C, the viability of the THP-1 macrophages treated with PDT (16 μg/mL UCNPs-Ce6 plus 60 seconds of 980 nm laser) declined significantly to 67%. However, the addition of pigskin restored cell viability decrease in a thickness-dependent manner, thereby illustrating that the tissues absorbed the 980 nm laser to a certain extent. The 980 nm laser penetrated the 6 mm pigskin and induced cell death. Wang et al22 confirmed that the efficiency of conventional PDT treatment using a 660 nm laser decreases by 80% if the laser is blocked by tissues with a thickness of 3 mm, whereas decreases of only 5% is noted in the setting of NIR-treated PDT. We also indirectly detected the accumulation of UCNPs-Ce6 in the THP-1 macrophages via the fluorescence of Ce6 (red color), and we observed that the fluorescence reached its highest level at 4 hours following incubation (Figure 1D).

Bottom Line: Our results indicated that the levels of ROS were significantly increased in the PDT group, resulting in both MPTP opening and MMP depolarization, which led to apoptosis.The proapoptotic factor Bax subsequently translocates from the cytosol to the mitochondria, resulting in the MPTP opening and cytochrome C release.This study demonstrated the great potential of UCNPs-Ce6-mediated PDT in the treatment of AS.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathophysiology, Harbin Medical University, Harbin, People's Republic of China.

ABSTRACT
Atherosclerosis (AS) is the most vital cardiovascular disease, which poses a great threat to human health. Macrophages play an important role in the progression of AS. Photodynamic therapy (PDT) has emerged as a useful therapeutic modality not only in the treatment of cancer but also in the treatment of AS. The purpose of this study was to determine the molecular mechanisms underlying the activity of PDT, using mesoporous-silica-coated upconversion fluorescent nanoparticles encapsulating chlorin e6 (UCNPs-Ce6) in the induction of apoptosis in THP-1 macrophages. Here, we investigated the ability of UCNPs-Ce6-mediated PDT to induce THP-1 macrophage apoptosis by facilitating the induction of reactive oxygen species (ROS) and regulation of mitochondrial permeability transition pore (MPTP) to depolarize mitochondrial membrane potential (MMP). Both Bax translocation and the release of cytochrome C were examined using immunofluorescence and Western blotting. Our results indicated that the levels of ROS were significantly increased in the PDT group, resulting in both MPTP opening and MMP depolarization, which led to apoptosis. In addition, immunofluorescence and Western blotting revealed that PDT induced both Bax translocation and the release of cytochrome C, as well as upregulation of cleaved caspase-9, cleaved caspase-3, and cleaved poly(ADP-ribose) polymerase. Therefore, we demonstrated that UCNPs-Ce6-mediated PDT induces apoptosis in THP-1 macrophages via ROS bursts. The proapoptotic factor Bax subsequently translocates from the cytosol to the mitochondria, resulting in the MPTP opening and cytochrome C release. This study demonstrated the great potential of UCNPs-Ce6-mediated PDT in the treatment of AS.

No MeSH data available.


Related in: MedlinePlus