Novel phthalocyanines activated by dim light for mosquito larva- and cell-inactivation with inference for their potential as broad-spectrum photodynamic insecticides.

Mosquitoes are significant vectors, responsible for transmitting serious infectious diseases, including the recent epidemics of global significance caused by, for example, Zika, Dengue and Chikungunya viruses. The chemical insecticides in use for mosquito control are toxic and ineffective due to the development of resistance to them. The new approach to reduce mosquito population by releasing genetically modified males to cause female infertility is still under environmental safety evaluation. Photodynamic insecticides (PDI) have long been known as a safe and effective alternative by using dyes as the photosensitizers (PS) for activation with light to generate insecticidal singlet oxygen and reactive oxygen species. This approach warrants re-examination with advances in the chemical synthesis of novel PS, e.g. phthalocyanines (PC). Nine PC were compared with five porphyrin derivatives and two classic PS of halogenated fluoresceins, i.e. cyanosine and rose bengal experimentally for photodynamic treatment (PDT) of the larvae of laboratory-reared Aedes mosquitoes and their cell lines. Groups of 2nd instar larvae were first exposed overnight to graded concentrations of each PS in the dark followed by their exposure to dim light for up to 7 hours. Larvae of both experimental and control groups were examined hourly for viability based on their motility. Monolayers of mosquito cells were similarly PS-sensitized and exposed briefly to light at the PS-specific excitation wavelengths. Cell viability was assessed by MTT reduction assays. Of the 16 PS examined for photodynamic inactivation of the mosquito larvae, effective are three novel PC, i.e. amino-Si-PC1 and -PC2, anilinium Zn-PC3.4, pyridyloxy Si-PC14 and two porphyrin derivatives, i.e. TPPS2 and TMAP. Their EC50 values were determined, all falling in the nanomolar range lower than those of rose bengal and cyanosine. All PS effective in vivo were also found to dose-dependently inactivate mosquito cells photodynamically in vitro, providing cellular basis for their larvicidal activities. The present findings of novel PC with effective photodynamic larvicidal activities provide fresh impetus to the development of PDI with their established advantages in safety and efficacy. Toward that end, the insect cell lines are of value for rapid screening of new PC. The optimal excitability of PC with insect-invisible red light is inferred to have the potential to broaden the range of targetable insect pests.

pH- and Thiol-Responsive BODIPY-Based Photosensitizers for Targeted Photodynamic Therapy.

A diiodo distyryl boron dipyrromethene (BODIPY) core was conjugated to two ferrocenyl quenchers through acid-labile ketal and/or thiol-cleavable disulfide linkers, of which the fluorescence and photosensitizing properties were significantly quenched through a photoinduced electron-transfer process. The two symmetrical analogues that contained either the ketal or disulfide linkers could only be activated by a single stimulus, whereas the unsymmetrical analogue was responsive to dual stimuli. Upon interaction with acid and/or dithiothreitol (DTT), these linkers were cleaved selectively. The separation of the BODIPY core and the ferrocenyl moieties restored the photoactivities of the former in phosphate buffered saline and inside the MCF-7 breast cancer cells, rendering these compounds as potential activable photosensitizers for targeted photodynamic therapy. The dual activable analogue exhibited the greatest enhancement in intracellular fluorescence intensity in both an acidic environment (pH 5) and the presence of DTT (4 mm). Its photocytotoxicity against MCF-7 cells also increased by about twofold upon preincubation with 4 mm of DTT. The activation of this compound was also demonstrated in nude mice bearing a HT29 human colorectal carcinoma. A significant increase in fluorescence intensity in the tumor was observed over 9 h after intratumoral injection.

Aminophthalocyanine-Mediated Photodynamic Inactivation of Leishmania tropica.

Photodynamic inactivation ofLeishmaniaspp. requires the cellular uptake of photosensitizers, e.g., endocytosis of silicon(IV)-phthalocyanines (PC) axially substituted with bulky ligands. We report here that when substituted with amino-containing ligands, the PCs (PC1 and PC2) were endocytosed and displayed improved potency againstLeishmania tropicapromastigotes and axenic amastigotesin vitro The uptake of these PCs by bothLeishmaniastages followed saturation kinetics, as expected. Sensitive assays were developed for assessing the photodynamic inactivation ofLeishmaniaspp. by rendering them fluorescent in two ways: transfecting promastigotes to express green fluorescent protein (GFP) and loading them with carboxyfluorescein succinimidyl ester (CFSE). PC-sensitizedLeishmania tropicastrains were seen microscopically to lose their motility, structural integrity, and GFP/CFSE fluorescence after exposure to red light (wavelength, ∼650 nm) at a fluence of 1 to 2 J cm(-2) Quantitative fluorescence assays based on the loss of GFP/CFSE from liveLeishmania tropicashowed that PC1 and PC2 dose dependently sensitized both stages for photoinactivation, consistent with the results of a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay.Leishmania tropicastrains are >100 times more sensitive than their host cells or macrophages to PC1- and PC2-mediated photoinactivation, judging from the estimated 50% effective concentrations (EC50s) of these cells. Axial substitution of the PC with amino groups instead of other ligands appears to increase its leishmanial photolytic activity by up to 40-fold. PC1 and PC2 are thus potentially useful for photodynamic therapy of leishmaniasis and for oxidative photoinactivation ofLeishmaniaspp. for use as vaccines or vaccine carriers.

Sequential logic operations with a molecular keypad lock with four inputs and dual fluorescence outputs.

A novel coumarin-rhodamine conjugate was prepared, and its metal binding properties were studied by UV/Vis and fluorescence spectroscopy. The conjugate serves as a ratiometric and highly selective fluorescent sensor for Hg(2+) ions. Its metal-responsive spectral properties were utilized to construct a molecular keypad lock with four inputs and dual fluorescence outputs. The complexity of this molecular logic network can greatly enhance the security level of this device.

A dual activatable photosensitizer toward targeted photodynamic therapy.

An unsymmetrical bisferrocenyl silicon(IV) phthalocyanine has been prepared in which the disulfide and hydrazone linkers can be cleaved by dithiothreitol and acid, respectively. The separation of the ferrocenyl quenchers and the phthalocyanine core greatly enhances the fluorescence emission, singlet oxygen production, intracellular fluorescence intensity, and in vitro photocytotoxicity. The results have been compared with those for the two symmetrical analogues which contain either the disulfide or hydrazone linker and therefore can only be activated by one of these stimuli. For the dual activatable agent, the greatest enhancement can be attained under a slightly acidic environment (pH = 4.5-6.8) and in the presence of dithiothreitol (in millimolar range), which can roughly mimic the acidic and reducing environment of tumor tissues. This compound can also be activated in tumor-bearing nude mice. It exhibits an increase in fluorescence intensity in the tumor over the first 10 h after intratumoral injection and can effectively inhibit the growth of tumor upon illumination.

[Spectral properties, photodynamic anticancer activity and the interaction with albumin of silicon phthalocyanine axially modified by pyrimidine derivatives].

Photophysical photochemical properties, in vitro photodynamic anticancer activity and interaction with albumin of a new axially modified silicon phthalocyanine, i. e. di (2-amino-6-trifluoromethyl-4-pyrimidinyloxy) silicon phthalocyanine (SiPcF), were studied in the present paper. The Q band maximum absorption of SiPcF located at 686 nm with the molar absorption coefficient of 2.3 x 10(5) mol(-1) * L * cm(-1). The fluorescence emission of SiPcF peaked at 694 nm with a fluorescence quantum yield of 0.46. Its singlet oxygen quantum yield produced by photosensitization is 0.38, suggesting that SiPcF is an effective 1O2 photosensitizer. There is strong interaction between SiPcF and bovine serum albumin (BSA). The binding constant is 4.33 x 10(5) mol X L(-1) and the number of binding sites is 1. In vitro experiments show that SiPcF had a high photodynamic anticancer activity against human hepatoma cells HepG2 with an IC50 value down to 5 x 10(-7) mol X L(-1).

A disulfide-linked conjugate of ferrocenyl chalcone and silicon(IV) phthalocyanine as an activatable photosensitiser.

A novel bis(ferrocenyl chalcone) silicon(IV) phthalocyanine has been prepared in which the disulfide linker can be cleaved by dithiothreitol. The separation of the ferrocenyl quencher and the phthalocyanine core greatly enhances the fluorescence emission, singlet oxygen production and in vitro photocytotoxicity.

A highly selective and sensitive BODIPY-based colourimetric and turn-on fluorescent sensor for Hg2+ ions.

A series of monostyryl boron dipyrromethenes appended with an NO(4), NO(2)S(2), N(3)O(4), or N(3)O(2)S(2)-type ligand have been prepared and characterised. While the UV-Vis spectra of the former three compounds in CH(3)CN/H(2)O (2 : 3 v/v) do not respond towards a wide range of metal ions, the derivative with an N(3)O(2)S(2)-ligand exhibits a highly selective and sensitive spectral response towards Hg(2+) ions. The absorption band is blue-shifted by 40 nm due to inhibition of the intramolecular charge transfer process upon metal complexation. The fluorescence is also turned on giving a strong emission band at 572 nm. The colour changes can be easily detected by the naked eye. The results suggest that this compound serves as a promising colourimetric and fluorescent sensor for Hg(2+) ions in this mixed aqueous medium.

Phthalocyanine-polyamine conjugates as highly efficient photosensitizers for photodynamic therapy.

A series of silicon(IV) phthalocyanines substituted axially with different polyamine moieties have been prepared. Their fluorescence quantum yields (Φ(F) = 0.03-0.08) in N,N-dimethylformamide are low because of reductive quenching by the amino moieties. The values are significantly increased in aqueous media (Φ(F) = 0.12-0.21) as a result of protonation of the amino substituents. All the compounds are highly photocytotoxic against human colon adenocarcinoma HT29 cells and Chinese hamster ovary cells with IC(50) values as low as 1.1 nM. Flow cytometric studies of two selected compounds (2 and 5) against HT29 cells have shown that they induce apoptosis extensively. As shown by confocal microscopy, these two compounds also show high affinity toward the lysosomes, but not the mitochondria, of the cells. Their in vivo photodynamic activity has also been investigated using HT29 tumor bearing nude mice. Both of them can effectively inhibit the growth of the tumor without causing apparent injury to the liver of the mice.

A pH-responsive fluorescence probe and photosensitiser based on a tetraamino silicon(IV) phthalocyanine.

A novel tetraamino silicon(iv) phthalocyanine has been prepared, of which the fluorescence emission and reactive oxygen species generation efficiency are greatly enhanced at lower pH in the range of ca. 5-7, making it a promising pH-controlled and tumour-selective fluorescence probe and photosensitiser for photodynamic therapy.

Phthalocyanine-polyamine conjugates as pH-controlled photosensitizers for photodynamic therapy.

A series of aryl hydroxyamines prepared by reductive amination were treated with silicon(IV) phthalocyanine dichloride in the presence of pyridine to give the diaxially substituted phthalocyanine-polyamine conjugates 1-5. The electronic absorption, fluorescence emission, and efficiency at generating reactive oxygen species of these compounds were all sensitive to the pH environment. Under acidic conditions, the fluorescence quantum yields and the singlet oxygen quantum yields of these compounds were greatly enhanced in DMF as a result of protonation of the amino moieties, which inhibited the photoinduced electron-transfer deactivation pathway. The Q band was diminished and broadened, and the fluorescence intensity decreased as the pH increased in citrate buffer solutions. The rate of superoxide radical formation was also reduced in a higher pH environment. Compound 3, containing a terminal 4-chlorophenyl group at the axial substituent, showed the most desirable pH-responsive properties, which makes it a promising tumor-selective fluorescence probe and photosensitizer for photodynamic therapy. All of the phthalocyanines 1-5 were highly photocytotoxic against HT29 and HepG2 cells with IC(50) values as low as 0.03 microM. Compound 3 was highly selective toward lysosomes, but not mitochondria of HT29 cells.

Synthesis and in vitro photodynamic activities of di-alpha-substituted zinc(ii) phthalocyanine derivatives.

A new series of di-alpha-substituted zinc(ii) phthalocyanine derivatives have been prepared by mixed cyclisation of the corresponding 1,4-disubstituted phthalonitriles or naphthalonitriles with an excess of unsubstituted phthalonitrile in the presence of Zn(OAc)(2).2H(2)O and 1,8-diazabicyclo[5.4.0]undec-7-ene. Having a large hydrophobic macrocyclic core substituted with two hydrophilic triethylene glycol chains or glycerol moieties, these compounds are amphiphilic in nature. They are highly soluble and remain non-aggregated in DMF as shown by the intense and sharp Q-band absorption. Compared with the unsubstituted zinc(ii) phthalocyanine, these di-alpha-substituted analogues exhibit a red-shifted Q band (at 689-701 nm), a relatively weaker fluorescence emission, and a higher efficiency to generate singlet oxygen. Upon illumination, these compounds are highly cytotoxic towards HT29 human colorectal carcinoma and HepG2 human hepatocarcinoma cells with IC(50) values as low as 0.06 muM. The high photocytotoxicity of these compounds can be attributed to their high cellular uptake and low aggregation tendency in the biological media, leading to a high efficiency to generate reactive oxygen species inside the cells.

Highly photocytotoxic 1,4-dipegylated zinc(II) phthalocyanines. Effects of the chain length on the in vitro photodynamic activities.

A new series of 1,4-dipegylated zinc(II) phthalocyanines have been synthesised and spectroscopically characterised. The derivatives ZnPc[O(CH(2)CH(2)O)(n)Me](2) (n = 2, 4, ca. 12) have been prepared by mixed cyclisation of the corresponding disubstituted phthalonitriles with an excess of unsubstituted phthalonitrile in the presence of Zn(OAc)(2) x 2 H(2)O and 1,8-diazabicyclo[5.4.0]undec-7-ene. The other two analogues ZnPc[O(CH(2)CH(2)O)(n)Me](2) (n = 6, 8) have been prepared by chain elongation reaction of ZnPc[O(CH(2)CH(2)O)(4)H](2). These macrocycles are highly soluble and remain non-aggregated in DMF as shown by the sharp Q-band absorption. Compared with the unsubstituted zinc(II) phthalocyanine, these di-alpha-substituted analogues have a red-shifted Q band (at 689 vs. 670 nm) and exhibit a relatively weaker fluorescence emission and a higher efficiency at generating singlet oxygen. Upon illumination, these compounds are highly cytotoxic toward HT29 human colorectal carcinoma and HepG2 human hepatocarcinoma cells. The compounds with medium-length substituents are particularly potent, having IC(50) values as low as 0.02 microM. The high photodynamic activity of these compounds can be attributed to their high cellular uptake and low aggregation tendency in the biological media, which promote the generation of reactive oxygen species inside the cells. The effects of the chain length on the aggregation behaviour, photophysical properties, cellular uptake and in vitro photodynamic activities of this series of compounds have also been examined.

Synthesis, Characterization, and In Vitro Photodynamic Activity of Novel Amphiphilic Zinc(II) Phthalocyanines Bearing Oxyethylene-Rich Substituents.

Three novel zinc(II) phthalocyanines substituted with one or two 3,4,5-tris(3,6,9-trioxadecoxy)benzoxy group(s) have been prepared and spectroscopically characterized. These compounds are highly soluble and remain nonaggregated in N,N-dimethylformamide. Upon excitation, they exhibit a relatively weak fluorescence emission and high efficiency to generate singlet oxygen compared with the unsubstituted zinc(II) phthalocyanine. These amphiphilic photosensitizers formulated with Cremophor EL are highly photocytotoxic against HT29 human colon adenocarcinoma and HepG2 human hepatocarcinoma cells. The mono-alpha-substituted analogue 4 is particularly potent with IC50 values as low as 0.02 muM. The higher photodynamic activity of this compound can be attributed to its lower aggregation tendency in the culture media as shown by absorption spectroscopy and higher cellular uptake as suggested by the stronger intracellular fluorescence, resulting in a higher efficiency to generate reactive oxygen species inside the cells.

Synthesis, photophysical properties and in vitro photodynamic activity of axially substituted subphthalocyanines.

A new series of subphthalocyanines substituted axially with an oligoethylene glycol chain [SPcB(OCH(2)CH(2))(n)OH, n = 3 (2) or 4 (3)] or a p-phenoxy oligoethylene glycol methyl ether chain [SPcBOC(6)H(4)(OCH(2)CH(2))(n)OCH(3), n = 2 (4) or 3 (5)] have been synthesised by substitution reactions of boron subphthalocyanine chloride SPcBCl (1) with the corresponding oligoethylene glycols, and characterised with various spectroscopic methods and elemental analysis. The molecular structure of one of these compounds (subphthalocyanine 4) has also been determined. As revealed by absorption spectroscopy, these compounds are essentially non-aggregated in DMF. The low aggregation tendency of these compounds results in a strong fluorescence emission and high efficiency to generate singlet oxygen. All these subphthalocyanines, being formulated with Cremophor EL, function as efficient photosensitisers and exhibit a high photocytotoxicity against HepG2 human hepatocarcinoma and HT29 human colon adenocarcinoma cells. The phenoxy analogues 4 and 5 show a relatively high photostability and are particularly potent towards these cell lines, with IC(50) values down to 0.02 microM.

Preparation and in vitro photodynamic activities of novel axially substituted silicon (IV) phthalocyanines and their bovine serum albumin conjugates.

Two novel axially substituted phthalocyanines, namely bis(4-(4-acetylpiperazine)phenoxy)phthalocyaninatosilicon (IV) (1) and its N-methylated derivative 2, have been synthesized. The dicationic phthalocyanine 2 is non-aggregated in water and exhibits good photophysical properties. The non-covalent BSA conjugates of these compounds have also been prepared. Compound 2 and the conjugate 2-BSA show extremely high photodynamic activities toward B16 melanoma cancer cell lines. The corresponding 50% growth-inhibitory (IC50) ratios are 33 and 38 nM, respectively.