The Relationship of Phthalocyanine 4 (Pc 4) Concentrations Measured Noninvasively to Outcome of Pc 4 Photodynamic Therapy in Mice

The ability to noninvasively measure photosensitizer concentration at target tissues will allow optimization of photodynamic therapy (PDT) and could improve outcome. In this study, we evaluated whether preirradiation tumor phthalocyanine 4 (Pc 4) concentrations, measured noninvasively by the optical pharmacokinetic system (OPS), correlated with tumor response to PDT. Mice bearing human breast cancer xenografts were treated with 2 mg kg⁻¹ Pc 4 iv only, laser irradiation (150 J cm⁻²) only, Pc 4 followed by fractionated irradiation or Pc 4 followed by continuous irradiation. Laser irradiation treatment was initiated when the tumor to skin ratio of Pc 4 concentration reached a maximum of 2.1 at 48 h after administration. Pc 4 concentrations in tumor, as well as in Intralipid in vitro , decreased monoexponentially with laser fluence. Pc 4-PDT resulted in significant tumor regression, and tumor response was similar in the groups receiving either fractionated or continuous irradiation treatment after Pc 4. Tumor growth delay following Pc 4-PDT correlated with OPS-measured tumor Pc 4 concentrations at 24 h prior to PDT ( R ² = 0.86). In excised tumors, OPS-measured Pc 4 concentrations were similar to the HPLC-measured concentrations. Thus, OPS measurements of photosensitizer concentrations can be used to assist in the scheduling of Pc 4-PDT.     

The peripheral benzodiazepine receptor in photodynamic therapy with the phthalocyanine photosensitizer Pc 4

The peripheral benzodiazepine receptor (PBR) is an 18 kDa protein of the outer mitochondrial membrane that interacts with the voltage-dependent anion channel and may participate in formation of the permeability transition pore. The physiological role of PBR is reflected in the high-affinity binding of endogenous ligands that are metabolites of both cholesterol and heme. Certain porphyrin precursors of heme can be photosensitizers for photodynamic therapy (PDT), which depends on visible light activation of porphyrin-related macrocycles. Because the apparent binding affinity of a series of porphyrin analogs for PBR paralleled their ability to photoinactivate cells, PBR has been proposed as the molecular target for porphyrin-derived photocytotoxicity. The phthalocyanine (Pc) photosensitizer Pc 4 accumulates in mitochondria and structurally resembles porphyrins. Therefore, we tested the relevance of PBR binding on Pc 4-PDT. Binding affinity was measured by competition with 3H-PK11195, a high-affinity ligand of PBR, for binding to rat kidney mitochondria (RKM) or intact Chinese hamster ovary (CHO) cells. To assess the binding of the Pc directly, we synthesized 14C-labeled Pc 4 and found that whereas Pc 4 was a competitive inhibitor of 3H-PK11195 binding to the PBR, PK11195 did not inhibit the binding of 14C-Pc 4 to RKM. Further, 14C-Pc 4 binding to RKM showed no evidence of saturation up to 10 microM. Finally, when Pc 4-loaded CHO cells were exposed to activating red light, apoptosis was induced; Pc 4-PDT was less effective in causing apoptosis in a companion cell line overexpressing the antiapoptotic protein Bcl-2. For both cell lines, PK11195 inhibited PDT-induced apoptosis; however, the inhibition was transient and did not extend to overall cell death, as determined by clonogenic assay. The results demonstrate (1) the presence of low-affinity binding sites for Pc 4 on PBR; (2) the presence of multiple binding sites for Pc 4 in RKM and CHO cells other than those that influence PK11195 binding; and (3) the ability of high supersaturating levels of PK11195 to transiently inhibit apoptosis initiated by Pc 4-PDT, with less influence on overall cell killing. We conclude that the binding of Pc 4 to PBR is less relevant to the photocytotoxicity of Pc 4-PDT than are other mitochondrial events, such as photodamage to Bcl-2 and that the observed inhibition of Pc 4-PDT-induced apoptosis by PK11195 likely occurs through a mechanism independent of PBR.     

荧光各向异性法研究酸度对四磺基铝酞菁与牛血清白蛋白相互作用的影响

对酞菁类化合物与白蛋白相互作用的研究有助于了解其在血液中的存在形式,从而探索其光敏作用机制[1].荧光各向异性法是研究小分子与蛋白质相互作用的有力工具[2].本文将荧光各向异性的原理和方法应用于由光敏剂四磺基铝酞菁(AlS4Pc)与牛血清白蛋白(BSA)组成的体系,考察了AlS4Pc的荧光各向异性随酸度变化的规律.结果表明,BSA对AlS4Pc有较强的结合能力,提示血清白蛋白对酞菁类光敏剂可起到储存和转运的作用.1　实验部分1.1　试剂及仪器　四磺基铝酞菁的合成与纯化参照文献[3]方法进行.产物经聚酰胺层析、紫外可见吸收光谱、荧光光谱、红…     

Dianionic Titanyl and Vanadyl (Cation+)2[MIVO(Pc4−)]2− Phthalocyanine Salts Containing Pc4− Macrocycles

In this study, the titanyl and vanadyl phthalocyanine (Pc) salts (Bu₄N⁺)₂[M^IVO(Pc^4?)]^2? (M=Ti, V) and (Bu₃MeP⁺)₂[M^IVO(Pc^4?)]^2? (M=Ti, V) with [M^IVO(Pc^4?)]^2? dianions were synthesized and characterized. Reduction of M^IVO(Pc^2?) carried out with an excess of sodium fluorenone ketyl in the presence of Bu₄N⁺ or Bu₃MeP⁺ is exclusive to the phthalocyanine centers, forming Pc^4? species. During reduction, the metal +4 charge did not change, implying that Pc is an non‐innocent ligand. The Pc negative charge increase caused the C?N(pyr) bonds to elongate and the C?N(imine) bonds to alternate, thus increasing the distortion of Pc. Jahn–Teller effects are significant in the [eg(π*)]² dianion ground state and can additionally distort the Pc macrocycles. Blueshifts of the Soret and Q‐bands were observed in the UV/Vis/NIR when M^IVO(Pc^2?) was reduced to [M^IVO(Pc ^{. 3?})] ^{. ?} and [M^IVO(Pc^4?)]^2?. From magnetic measurements, [Ti^IVO(Pc^4?)]^2? was found to be diamagnetic and (Bu₄N⁺)₂[V^IVO(Pc^4?)]^2? and (Bu₃MeP⁺)₂[V^IVO(Pc^4?)]^2? were found to have magnetic moments of 1.72–1.78 μ_B corresponding to an S=1/2 spin state owing to V^IV electron spin. As a result, two latter salts show EPR signals with V^IV hyperfine coupling.     

Potentiation of photodynamic therapy in mice with recombinant human tumor necrosis factor-alpha.

The tumoricidal response of subcutaneously growing SMT-F adenocarcinoma implanted into syngeneic DBA/2 mice to Photofrin II-sensitized photodynamic therapy (PDT) was statistically significantly enhanced by the addition of a single dose of intravenously administered recombinant human tumor necrosis factor-alpha (rHuTNF-alpha). The interaction appeared to be approximately additive, i.e. tumor response to PDT plus rHuTNF-alpha was about the same as that observed by doubling the PDT dose. Conversely, rHuTNF-alpha did not significantly potentiate the cutaneous phototoxicity in mouse feet due to PDT. These data suggest that combination therapy should be considered for improving tumor response while retaining treatment selectivity in human malignancies.     

Antivascular tumor eradication by hypericin-mediated photodynamic therapy

Photodynamic therapy (PDT) with hypericin has been shown to inhibit tumor growth in different tumor models, and tumor vascular damage was suggested to be mainly responsible for the antitumoral effect. Here, we demonstrate tumor vascular damage and its consequence on local tumor control after hypericin-mediated PDT by using both short and long drug-light intervals. Radiation-induced fibrosarcoma-1 tumors were exposed to laser light at either 0.5 or 6 h after a 5 mg/kg dose of hypericin. Tumor perfusion was monitored by fluorescein dye-exclusion assay and by Hoechst 33342 staining of functional blood vessels. Significant reduction in tumor perfusion was found immediately after both PDT treatments. A complete arrest of vascular perfusion was detected by 15 h after the 0.5 h-interval PDT, whereas well-perfused areas could still be found at this time in tumors after the 6 h-interval PDT. A histological study confirmed that primary vascular damage was involved in both PDT treatments. Tumor cells appeared intact shortly after light treatment, degenerated at later hours and became extensively pycnotic at 24 h after the 0.5 h-interval PDT. PDT under this condition led to complete tumor cure. In contrast, significant numbers of viable tumor cells, especially at the tumor periphery, were found histologically at 24 h after the 6 h-interval PDT. No tumor cure was obtained when PDT was performed at this time. Our results strongly suggest that targeting the tumor vasculature by applying short drug-light interval PDT with hypericin might be a promising way to eradicate solid tumors.     

Diketopyrrolopyrrole-Au(I) as singlet oxygen generator for enhanced tumor photodynamic and photothermal therapy

Diketopyrrolopyrrole(DPP) derivatived photosensitizers(PSs) with near infrared(NIR) absorption and good photophysical properties have drawn tremendous attention in cancer phototherapy. However, current DPP derivatives present unsatisfactory quantum yield of singlet oxygen(~1O_2) due to the large energy gap between the excited singlet and triplet states. To tackle this challenge, herein the DPP core is functionalized with triphenylphosphine-Au(I) group(Th DPP-Au), leading to a high~1O_2 quantum yield of 0.65. Theoretical calculation attributes the enhancement to spin-orbit coupling and population of the triplet excition upon photoexcitation. The hydrophilic Th DPP-Au nanoparticals(NPs) are prepared via nano-reprecipitation, which displays homogeneous size and excellent light absorption ability(ε=4.382×10~4 M~(-1)cm~(-1)). And the Th DPP-Au NPs exhibit low dark toxicity and high phototoxicity, which can effectively kill tumor cells via ~1O_2 induced mitochondrial apoptotic pathway upon irradiation. Furthermore, in vivo experiments demonstrate that Th DPP-Au NPs can selective accumulation in tumor and present excellent synergistic photodynamic/photothermal therapy guided by fluorescence and photothermal dual imaging.     

Photodynamic therapy with Pc 4 induces apoptosis of Candida albicans

The high prevalence of drug resistance necessitates the development of novel antifungal agents against infections caused by opportunistic fungal pathogens, such as Candida albicans. Elucidation of apoptosis in yeast-like fungi may provide a basis for future therapies. In mammalian cells, photodynamic therapy (PDT) has been demonstrated to generate reactive oxygen species, leading to immediate oxidative modifications of biological molecules and resulting in apoptotic cell death. In this report, we assess the in vitro cytotoxicity and mechanism of PDT, using the photosensitizer Pc 4, in planktonic C. albicans. Confocal image analysis confirmed that Pc 4 localizes to cytosolic organelles, including mitochondria. A colony formation assay showed that 1.0 μM Pc 4 followed by light at 2.0 J cm(-2) reduced cell survival by 4 logs. XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide) assay revealed that Pc 4-PDT impaired fungal metabolic activity, which was confirmed using the FUN-1 (2-chloro-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenylquinolinium iodide) fluorescence probe. Furthermore, we observed changes in nuclear morphology characteristic of apoptosis, which were substantiated by increased externalization of phosphatidylserine and DNA fragmentation following Pc 4-PDT. These data indicate that Pc 4-PDT can induce apoptosis in C. albicans. Therefore, a better understanding of the process will be helpful, as PDT may become a useful treatment option for candidiasis.     

Mechanisms of tumor necrosis induced by photodynamic therapy

Despite great progress and promising results achieved in cancer treatment by photodynamic therapy (PDT), the exact mechanism of tumor photosensitization in vivo by porphyrins and related phototherapeutic agents has not been fully explored and understood. This review is an attempt to gather available data on various processes occurring in neoplastic cells, microvasculature, non-vascular stroma and circulating blood within PDT-treated tumors. This information is necessary to understand the mechanisms governing the very complex processes which eventually lead to tumor necrosis.     

DH-I-180-3-mediated photodynamic therapy: biodistribution and tumor vascular damage

An important goal of photodynamic therapy (PDT) for treatment of various cancers is to shorten PDT-performing time and simultaneously enhance PDT efficacy. Here, we investigated the nontumor tissue distribution of and the tumor vascular damage caused by a new photosensitizer, DH-I-180-3, in mice with implanted EMT6 mammary tumor cells. In addition, we performed cell-based assays to evaluate the basic antitumor effect of DH-I-180-3/PDT in EMT6 cells. After administration of PDT, the type of cell death was characterized to be apoptosis, and a change in the mitochondrial membrane potential was also observed within minutes. On the other hand, tumor growth was remarkably retarded in vivo in mice that received DH-I-180-3/PDT, compared with mice in the control group, which were exposed to light irradiation alone. Finally, tumors in some mice nearly healed. The antitumor drug reached a maximum concentration approximately 3 h after administration. However, PDT was most effective when there was substantial accumulation of DH-I-180-3 in the tumor vasculature and in healthy tissue. The histological demonstration provided further evidence of tumor vascular damage. On the basis of these findings, we suggest that PDT with the photosensitizer DH-I-180-3 induces vascular damage with blood vessel shutdown, in addition to direct killing of tumor cells, in mice.     

Near-infrared light (NIR)-responsive nanoliposomes combining photodynamic therapy and chemotherapy for breast tumor control

Light-responsive carriers have been used for the controlled release of antitumor drugs in recent years. However, most light-responsive vectors require high-energy ultraviolet or visible light to achieve local drug release, and ultraviolet light would cause cellular damage. Near-infrared light has a deeper tissue-penetration depths and minimal harm to tissues, but it is difficult to cleave the chemical bond directly. The aim of this study is to develop a novel near-infrared light-responsive carrier for local release of antitumor drugs. Unsaturated phospholipids can be oxidized by singlet oxygen to achieve liposomal drug release, and singlet oxygen can be produced by photosensitizer under light irradiation. A new near-infrared light-responsive nanoliposome was designed that imparts light-triggered local drug release. Nanoliposomes, which were composed of matrix phospholipids and unsaturated phospholipids, were prepared by ammonium sulfate gradient method, and loaded with antitumor drug doxorubicin (DOX) and photosensitizer 1,4,8,11,15,18,22,25-octabutoxypalladium phthalocyanine. Under near-infrared light, photosensitizers could produce singlet oxygen and damage tumor cells by photodynamic therapy. Simultaneously, the unsaturated phospholipids were oxidized by singlet oxygen and result in DOX release, causing sustained cell damage by chemotherapy. Near-infrared light-responsive nanoliposomes exhibit enhanced anticancer activity owing to combined treatment of photodynamic therapy and chemotherapy. A new platform is thus offered for designing effective intracellular drug-release systems, holding great promise for future cancer therapy.     

Improvement of tumor response by manipulation of tumor oxygenation during photodynamic therapy

Photodynamic therapy (PDT) requires molecular oxygen during light irradiation to generate reactive oxygen species. Tumor hypoxia, either preexisting or induced by PDT, can severely hamper the effectiveness of PDT. Lowering the light irradiation dose rate or fractionating a light dose may improve cell kill of PDT-induced hypoxic cells but will have no effect on preexisting hypoxic cells. In this study hyperoxygenation technique was used during PDT to overcome hypoxia. C3H mice with transplanted mammary carcinoma tumors were injected with 12.5 mg/kg Photofrin and irradiated with 630 nm laser light 24 h later. Tumor oxygenation was manipulated by subjecting the animals to 3 atp (atmospheric pressure) hyperbaric oxygen or normobaric oxygen during PDT light irradiation. The results show a significant improvement in tumor response when PDT was delivered during hyperoxygenation. With hyperoxygenation up to 80% of treated tumors showed no regrowth after 60 days. In comparison, when animals breathed room air, only 20% of treated tumors did not regrow. To explore the effect of hyperoxygenation on tumor oxygenation, tumor partial oxygen pressure was measured with microelectrodes positioned in preexisting hypoxic regions before and during the PDT. The results show that hyperoxygenation may oxygenate preexisting hypoxic cells and compensate for oxygen depletion induced by PDT light irradiation. In conclusion, hyperoxygenation may provide effective ways to improve PDT efficiency by oxygenating both preexisting and treatment-induced cell hypoxia.     

Apoptosis mechanisms related to the increased sensitivity of Jurkat T-cells vs A431 epidermoid cells to photodynamic therapy with the phthalocyanine Pc 4

To examine the clinical applicability of Pc 4, a promising second-generation photosensitizer, for the photodynamic treatment of lymphocyte-mediated skin diseases, we studied the A431 and Jurkat cell lines, commonly used as surrogates for human keratinocyte-derived carcinomas and lymphocytes, respectively. As revealed by ethyl acetate extraction and absorption spectrophotometry, uptake of Pc 4 into the two cell lines was linear with Pc 4 concentration and similar on a per cell basis but greater in Jurkat cells on a per mass basis. Flow cytometry showed that uptake was linear at low doses; variations in the dose-response for uptake measured by fluorescence supported differential aggregation of Pc 4 in the two cell types. As detected by confocal microscopy, Pc 4 localized to mitochondria and endoplasmic reticulum in both cell lines. Jurkat cells were much more sensitive to the lethal effects of phthalocyanine photodynamic therapy (Pc 4-PDT) than were A431 cells, as measured by a tetrazolium dye reduction assay, and more readily underwent morphological apoptosis. In a search for molecular factors to explain the greater photosensitivity of Jurkat cells, the fate of important Bcl-2 family members was monitored. Jurkat cells were more sensitive to the induction of immediate photodamage to Bcl-2, but the difference was insufficient to account fully for their greater sensitivity. The antiapoptotic protein Mcl-1 was extensively cleaved in a dose- and caspase-dependent manner in Jurkat, but not in A431, cells exposed to Pc 4-PDT. Thus, the greater killing by Pc 4-PDT in Jurkat compared with A431 cells correlated with greater Bcl-2 photodamage and more strongly to the more extensive Mcl-1 degradation. Pc 4-PDT may offer therapeutic advantages in targeting inflammatory cells over normal keratinocytes in the treatment of T-cell-mediated skin diseases, such as cutaneous lymphomas, dermatitis, lichenoid tissue reactions and psoriasis, and it will be instructive to evaluate the role of Bcl-2 family proteins, especially Mcl-1, in the therapeutic response.     

Combination of phosphatidylinositol 3-kinases pathway inhibitor and photodynamic therapy in endothelial and tumor cells

Tumor recurrence due to incomplete eradication of tumor cells is a major problem facing current cancer therapies. To overcome this problem, it is necessary to enhance cell killing and/or prevent cell regrowth after treatment. Because phosphatidylinositol 3-kinases (PI3K) pathway plays an important role in stimulating cell survival and growth, we studied the feasibility of using a PI3K pathway inhibitor NVP-BEZ235 (BEZ235) to enhance the effectiveness of vascular-targeted photodynamic therapy (vPDT) with verteporfin. We found that BEZ235 or PDT alone significantly inhibited cell growth in both SVEC endothelial and PC-3 prostate cancer cells, although SVEC cells appeared to be more responsive than PC-3 cells. Autophagy was detected after both BEZ235 and verteporfin-PDT in both cell lines. Autophagy appeared to protect cells from PDT-induced cell death because inhibition of autophagy increased cell death. Autophagic flux assay revealed that PDT actually decreased autophagic flux especially at a high dose of verteporfin. Combination of BEZ235 and PDT caused greater inhibition of PI3K signaling pathway, leading to enhanced cell growth inhibition in both cell lines. SVEC cells exhibited a higher sensitivity towards such a combination than PC-3 cells. Our data indicated that BEZ235 in combination with PDT provides a promising approach of enhancing therapeutic response.     

Hyperoxygenation enhances the tumor cell killing of photofrin-mediated photodynamic therapy

Tumor hypoxia, either preexisting or as a result of oxygen depletion during photodynamic therapy (PDT) light irradiation, can significantly reduce the effectiveness of PDT-induced cell killing. To overcome tumor hypoxia and improve tumor cell killing, we propose using supplemental hyperoxygenation during Photofrin-PDT. The mechanism for the tumor cure enhancement of the hyperoxygenation-PDT combination is investigated using an in vivo-in vitro technique. A hypoxic tumor model was established by implanting mammary adenocarcinoma in the hind legs of mice. Light irradiation (200 J/cm2 at either 75 or 150 mW/cm2), under various oxygen supplemental conditions (room air, carbogen, 100% normobaric or hyperbaric oxygen), was delivered to animals that received 12.5 mg/kg Photofrin 24 h before light irradiation. Tumors were harvested at various time points after PDT and grown in vitro for colony formation analysis. Treated tumors were also analyzed histologically. The results show that when PDT is combined with hyperoxygenation, the hypoxic condition could be improved and the cell killing rate at various time points after PDT could be significantly enhanced over that without hyperoxygenation, suggesting an enhanced direct and indirect cell killing associated with high-concentration oxygen breathing. This study further confirms our earlier observation that when a PDT treatment is combined with hyperoxygenation it can be more effective in controlling hypoxic tumors.     

131I-labeled 5,10,15,20-tetrakis(4-hydroxyphenyl)porphyrin and 5,10,15,20-tetrakis(4-aminophenyl)porphyrin for combined photodynamic and radionuclide therapy

Journal of Radioanalytical and Nuclear Chemistry - Porphyrin derivatives are usually used for photodynamic therapy (PDT) and labeled with raidonuclide for radiopharmaceuticals. In this work,...     

Metal phthalocyanine as photosensitizer for photodynamic therapy (PDT)

A series of sulfonated (S) phthalimidomethyl (P) zinc phthalocyanines (Pc) was synthesized in a reaction, in which both kinds of substituents were introduced to ZnPc simultaneously. The products were separated by HPLC. The five different fractions obtained were further purified by a membrane separation method, and then characterized by UV/Vis, IR, element analysis and the abilities to generate singlet oxygen upon irradiation by light as well as a preliminary determination ofin vitro antitumor activities. The results show that one of the five separating parts with formula of ZnPcS₂P₂ exhibited rather good PDT activity. The compound was further characterized by NMR, MS and thermal analysis. Studies onin vivo antitumor activities of ZnPcS₂P₂ as photosensitizer show that its inhibitory rate was up to 89.8% and 90.8% for S₁₈₀ and U₁₄ solid tumors transplanted in mice respectively when the dosage of drug was 2 mg/kg and the dosage of laser light with 670 nm wavelength was 72 J/cm². Several structural factors influenced on the PDT activities were discussed.     

Effect of meta-tetra(hydroxyphenyl)chlorin (mTHPC)-mediated photodynamic therapy on sensitive and multidrug-resistant human breast cancer cells

Meta-tetra(hydroxyphenyl)chlorin (mTHPC) is in clinical trials for the photodynamic therapy (PDT) of localized-stage cancer. The PDT susceptibility of cells expressing multidrug resistance (MDR) phenotype is an attractive possibility to overcome the resistance to cytotoxic drugs observed during cancer chemotherapy. The accumulation, photocytotoxicity and intracellular localization of mTHPC were examined using the doxorubicin selected MCF-7/DXR human breast cancer cells, expressing P-glycoprotein (P-gp), and the wild-type parental cell line, MCF-7. No significant difference in mTHPC accumulation was observed between the two cell lines up to 3 h contact. The photodynamic activity of mTHPC, measured 24 h after irradiation with red laser light (lambda=650 nm), was significantly greater in MCF-7/DXR as compared to MCF-7 cells. A light dose of 2.5 J cm(-2) inducing 50% of cytotoxicity in MCF-7, resulted in 85% cytotoxicity in MCF-7/DXR. The presence of P-gp inhibitors SDZ-PSC-833 and cyclosporin A did not modify the mTHPC-induced cytotoxicity. The difference in intracellular mTHPC distribution pattern between two cell lines may contribute to different photocytotoxicity. Our results indicate that mTHPC mediated PDT could be useful in killing cells expressing MDR phenotype.