Synthesis and evaluation of novel fluorinated hematoporphyrin ether derivatives for photodynamic therapy.

A photosensitizer with high phototoxicity, suitable amphipathy and low dark toxicity could play a pivotal role in photodynamic therapy (PDT). In this study, a facile and versatile approach was adopted to synthesize a series of novel fluorinated hematoporphyrin ether derivatives (I1-I5 and II1-II4), and the photodynamic activities of these compounds were studied. Compared to hematoporphyrin monomethyl ether (HMME), all PSs showed preferable photodynamic activity against A549 lung tumor cells. The longest visible absorption wavelength of these compounds was approximately 622 nm. Among them, II3 revealed the highest singlet oxygen yield (0.0957 min-1), the strongest phototoxicity (IC50 = 1.24 µM), the lowest dark toxicity in vitro, and exhibited excellent anti-tumor effects in vivo. So compound II3 could act as new drug candidate for photodynamic therapy.

Luminescence and photosensitivity of gadolinium labeled hematoporphyrin monomethyl ether.

Photodynamic therapy for deep-lying lesions needs an appropriate imaging modality, precise evaluation of tissue oxygen and an effective photosensitizer. Gadolinium based metalloporphyrins Gd(III)-HMME is proposed in this study as a potential multifunctional theranostic agent, as photosensitizer, ratiometric oxygen sensor and MRI contrast agent. The time resolved spectroscopy revealed the luminescence peak of Gd(III)-HMME at 710 and 779 nm with a lifetime of 64 µs in oxygen-free methanol to be phosphorescent. This phosphorescence is strongly dependent on dissolved oxygen concentration. Its intensity in oxygen saturated methanol solution is 21% of that in deoxygenated solution. The singlet oxygen quantum yields ΦΔ of HMME and Gd(III)-HMME in air saturated methanol solution were determined to be 0.79 and 0.40 respectively using comparative spectra method. These phenomena indicate that the oxygen sensibility and production of singlet oxygen of Gd(III)-HMME can fulfill the requirement of PDT treatment.

A new antibacterial nano-system based on hematoporphyrin-carboxymethyl chitosan conjugate for enhanced photostability and photodynamic activity.

To promote bactericidal activity, improve photostability and safety, novel antibacterial nanoparticle system based on photodynamic action (PDA) was prepared here through conjugation of photosensitizer hematoporphyrin (HP) onto carboxymethyl chitosan (CMCS) via amide linkage and followed by ultrasonic treatment. The system was stable in PBS (pH 7.4) and could effectively inhibit the photodegradation of conjugated HP because of aggregation-caused quenching effect. ROS produced by the conjugated HP under light exposure could change the structure of nanoparticles by oxidizing the CMCS skeleton and thereby significantly promote the photodynamic activity of HP and its photodynamic activity after 6 h was higher than that of HP·2HCl under the same conditions. Antibacterial experiments showed that CMCS-HP nanoparticles had excellent photodynamic antibacterial activity, and the bacterial inhibition rates after 60 min of light exposure were greater than 97%. Safety evaluation exhibited that the nanoparticles were safe to mammalian cells, showing great potential for antibacterial therapy.

[Effect of ultrasound activating hematoporphyrin on the activities of antioxidative enzymes in mouse hepatoma 22].

This investigation was made with regard to the influences of ultrasound combined with hematoporphyrin on the activities of antioxidative enzyme in ascites hepatoma 22 (H-22) tumor cells, and to a better understanding of the potential biological mechanism of sonodynamic therapy which involved the damage to cells. Combined with 100 microg/ml hematoporphyrin, high intensity focused ultrasound sonication at a frequency of 1.43 MHz and an intensity level of 2.0 W/cm2 was delivered to H-22 tumor cells for 1 min. The viability of cells was evaluated by typan-blue blue exclusion test. The intracellular reactive oxygen species (ROS) levels were determined by 2',7'-dichlorofluorescein diacetata (DCFH-DA). Enzymatic chemical methods were used to measure the activities of key antioxidative enzymes. The results indicated that the cell damage rate of ultrasound combined with hematoporphyrin was significantly higher than that of the treatment with ultrasound alone, and hematoporphyrin alone had no killing effect on H-22 cells. The level of ROS in cell suspension was significantly increased, and the key antioxidative enzyme activities were obviously decreased after treatment with the combined use of ultrasound and hematoporphyrin. We speculated that the decreased activities of key antioxidative enzymes in cells might be involved in mediating the killing effect on H22 cells in sonodynamic therapy.

Towards environmentally and human friendly insect pest control technologies: photosensitization of leafminer flies Liriomyza bryoniae.

Development of new, ecologically safe technologies to control insect pest populations is of great importance. Photoactive compounds usually used for photosensitization might be effective as pesticide agents, with low impact on the environment, being non-toxic and not mutagenic. Phosensitizer accumulates within the insect body and, following exposure to visible light, induces lethal photochemical reactions and death. The aim of this study is to evaluate the possible usage of several photosensitizers (acridine orange, aminolevulinic acid, hematoporphyrin dimethyl ether, methylene blue) as photopesticides to control population of polyphagous plant pest Liriomyza bryoniae (Kaltenbach, 1858) (Diptera, Agromyzidae). Fluorescence measurements of intact cooled insects indicate that insect feeding with bait containing HPde and sugar induces remarkable accumulation of this compound in the body of insect. This accumulation is strongly dependent on sex and feeding duration. The highest HPde amount in the body of insect was detected 16 h after feeding, whereas no significant photosensitizer amount was detected in the same insect following 48 h. Following irradiation with visible light results in fast death of L. bryoniae. Of importance to note that survival of insects after feeding and irradiation depends on sex: female insect died much faster than males.

Energetics and efficiency of photoinactivation of murine tumor cells containing hematoporphyrin.

Hematoporphryrin derivative at an intracellular concentration in TA-3 mouse mammary carcinoma cells of 0.6 or 0.9 mM required input of 3.0 X 3.6 X 10(9) quanta/cell of red light (620 nm) to achieve a 90% kill. At an intracellular concentration of 1.2 mM, this light requirement drops to 1.5 X 10(9)quanta/cell. The energy for this photodynamic process is about 100 times higher than that required for ionizing radiation to achieve the same level of kill for these cells. The quantum yield for singlet oxygen formation (the cytotoxic agent in most photodynamic processes) from hematoporphyrin derivative is 0.75 +/- 0.07 in ethanol but only 0.16 +/- 0.07 within TA-3 cells, indicating possible intracellular complexing and quenching.

The photodynamic modification of DNA by hematoporphyrin.

Illumination (white light: 300-750 nm) of DNA in the presence of hematoporphyrin (less than or equal to 5-10 (-4) M) results in selective degradation of the guanine moiety. DNA so illuminated exhibits physical chemical properties (lowered sedimentation coefficients, lower temperatures of helix-coil transitions, increased buoyant density values) consistent with single-chain scissions (and the generation of single-stranded regions) which presumably are secondary to the photodegradation of the guanine residue. Illumination of DNA in the presence of low levels of hematoporphyrin (greater than or equal to 2.5 - 10 (-4) M) results in a biopolymer exhibiting all of the physical properties described above with the exception of a lowered sedimentation coefficient; on the contrary such DNA is aggregated. Of the four usual deoxynucleosides irradiated in the presence of hematoporphyrin, only deoxyguanosine is destroyed.

The effect of pH on the topography of porphyrins in lipid membranes.

The effect of the acidity of the environment on the topography and photophysics of sensitizer molecules in homogeneous solutions, and when embedded in a lipid microenvironment, was studied. Four hematoporphyrin (HP) analogs were studied, which have chemical "spacers" of varying lengths between the chromophoric tetrapyrrole and the carboxylate moiety. These derivatives have essentially the same chemical attributes and reactivity as the parent compound, HP IX, which is used in clinical procedures of photodynamic therapy. The binding constants of these HP derivatives to membrane model systems increase with the length of carboxylate chain in the pH range 3.0-6.6. This effect of chain length is attributed to an increase in the hydrophobicity of the molecule on elongation of the alkyl chains. A strong pH dependence of the quenching efficiency of the porphyrins' fluorescence by iodide ions was observed in aqueous solution and is attributed to a unique electrostatic interaction between the fluorophore and the quencher. The quenching efficiency in liposomes, relative to the quenching in buffer, as a function of pH, shows that porphyrins in the neutral form penetrate deeper inside the lipid bilayer and are less exposed to external quenching than when negatively charged at the carboxylic moiety. This vertical displacement in the membrane is also evidenced in the effect of pH on the photosensitized oxidation efficiency of a membrane-bound chemical target. Increasing the pH causes a significant decrease in the sensitization efficiency in liposomes. This trend is attributed to the vertical localization, and protonation of the carboxylic groups on lowering the pH leads to sinking of the sensitizer into the lipid bilayer and to a consequent generation of singlet oxygen at a deeper point. This increases the dwell time of singlet oxygen within the bilayer, which results in greater photodamage to a membrane-residing singlet oxygen target.

Neurovascular effects of reactive oxygen intermediates produced by photoradiation.

These experiments examined the effect of reactive oxygen intermediates, produced by laser illumination of the photosensitizer hematoporphyrin derivative, on the accumulation and release of norepinephrine from sympathetic nerve terminals. Using an isolated, spirally cut, superfused caudal artery of the rat, basal overflow of norepinephrine (NE) was significantly increased both during and after generation of reactive oxygen intermediates. Generation of reactive oxygen intermediates increased overflow of NE in vascular preparations in which release of NE had previously been elevated by the continuous superfusion of Krebs' solution, containing high concentrations of potassium (40 mM). Calcium free solutions did not block the overflow of norepinephrine augmented by reactive oxygen intermediates. This increase in overflow was due both to an increase in release of NE and an inhibition of accumulation of NE.

Effect of bleaching of porphyrin sensitizers during photodynamic therapy.

Cells of the line NHIK 3025 were incubated with the tumor-localizing porphyrin preparation DHE (Photofrin II). Exposure of light within the porphyrin absorption spectrum resulted in a change of the fluorescence spectra indicative of a movement of porphyrins from binding sites on proteins to more lipid environments, a degradation of the porphyrins in the cells without any significant formation of new porphyrin species. The photodegradation of the porphyrins resulted in a reduced yield of inactivation of cells per incident photon. Several independent estimations indicate that bleaching may play a role in clinical situations.

Hydrogen peroxide and hydroxyl free radical production by hematoporphyrin derivative, ascorbate and light.

Photofrin II, a commercial preparation of a hematoporphyrin derivative containing a high proportion of the active di-hematoporphyrin ether, was found to convert oxygen to hydrogen peroxide and hydroxyl free radicals in the presence of ascorbate and light. In addition, a light-dependent increase in the ascorbate free radical concentration was observed in hematoporphyrin derivative/ascorbate solutions with subsequent oxidation of ascorbate.

Photodynamic inactivation of simian immunodeficiency virus.

A photodynamic flow system employing a dihematoporphyrin ether (DHE) was tested for its ability to inactivate the in vitro infectivity of simian immunodeficiency virus (SICMac) at 630 +/- 5 nm with a light fluence of 5 J/cm2. Cell-free SIVMac was inactivated by photoactivated hematoporphyrin derivative in a dose-dependent fashion. Since SIVMac is closely related to human immunodeficiency virus type 2 (HIV-2) and we have previously reported the successful photodynamic inactivation of HIV-1 in cell-free medium as well as in whole human blood, this technology has the potential for the eradication of transfusion-associated acquired immunodeficiency diseases caused by the above-mentioned retroviruses.

The photodegradation of porphyrins in cells can be used to estimate the lifetime of singlet oxygen.

NHIK 3025 cells were incubated with Photofrin II (PII) and/or tetra (3-hydroxyphenyl)porphyrin (3THPP) and exposed to light at either 400 or 420 nm, i.e. at the wavelengths of the maxima of the fluorescence excitation spectra of the two dyes. The kinetics of the photodegradation of the dyes were studied. When present separately in the cells the two dyes are photodegraded with a similar quantum yield. 3THPP is degraded 3-6 times more efficiently by light quanta absorbed by the fluorescent fraction of 3THPP than by light quanta absorbed by the fluorescent fraction of PII present in the same cells. The distance diffused by the reactive intermediate, supposedly mainly 1O2, causing the photodegradation was estimated to be on the order of 0.01-0.02 micron, which corresponds to a lifetime of 0.01-0.04 microsecond of the intermediate in the cells. PII has binding sites at proteins in the cells as shown by an energy transfer band in the fluorescence excitation spectrum at 290 nm. During light exposure this band decays faster than the Soret band of PII under the present conditions. Photoproducts (1O2 etc.) generated at one binding site contribute significantly in the destruction of remote binding sites.

Radiation-induced formation of haematoporphyrin-transients in aqueous solution. A pulse radiolysis study.

The radiation-induced formation of some haematoporphyrin (HP) transients in aqueous solution was studied by pulse radiolysis. The OH radicals attack HP (rate constant k = (1.3 +/- 0.2) x 10(10) dm3 mol-1 s-1) and the resulting transients decay with 2k = (2.5 +/- 0.2) x 10(8) dm3 mol-1 s-1. The spectrum of the HP-OH species shows several maxima in the range of 450-850 nm (epsilon 590 = 3880, epsilon 640 = 3850, epsilon 800 = 3670 dm3 mol-1 cm-1). k for one electron reduction of HP (radical anion, HP.-) was determined by a direct eaq- attack on HP (at pH 9.0, k = (1.6 +/- 0.2) x 10(10) dm3 mol-1 s-1) as well as by electron transfer from hydroxyisopropyl radicals to the substrate (at pH 8.6, k = (3.2 +/- 0.1) x 10(8), at pH 3.8, k = (4.0 +/- 0.2) x 10(8), and at pH 2.0, k = (2.1 +/- 0.1) x 10(9) dm3 mol-1 s-1). In slightly alkaline media (pH 8.6 - 9.5) the HP.- transients have main absorption bands at lambda < 450 and 640 nm (epsilon 660 = 7500 dm3 mol-1 cm-1), whereas at pH 2.0 they are at lambda < 430 and 640 nm (epsilon 640 = 8170 dm3 mol-1 cm-1) and 710 nm (epsilon 710 = 8500 dm3 mol-1 cm-1). The one-electron oxidation of HP (formation of HP.+, radical cation) was studied by means of N3. or SO4.- species as electron acceptors. Using N3. at pH 9.5 k (HP + N3.) = (3.2 +/- 0.2) x 10(9) and for the decay 2k = (3.5 x 0.3) x 10(8) dm3 mol-1 s-1 were determined; at pH 3.6, k = (HP + N3.) = (7.1 +/- 0.3) x 10(8) and 2k = (2.6 +/- 0.3) x 10(7) dm3 mol-1 s-1 were found. In the case of SO4.- at pH9.8 a buildup (5.6 +/- 0.3) x 10.7 dm3 mol-1 s-1) was obtained. The spectrum of the HP.+ transients at pH 9.6 possesses several maxima with epsilon 345 = 38,800, epsilon 450 = 2400, epsilon 595 = 2880, and epsilon 660 = 3300 dm3 mol-1 cm-1. The corresponding values at pH 3.8 are epsilon 640 = 2450, epsilon 710 = 2600, and epsilon 780 = 1580 dm3 mol-1 cm-1.(ABSTRACT TRUNCATED AT 400 WORDS)

Solvents effects in the photodegradation and reactivity of the various ionic forms of haematoporphyrin.

The dependence of the ionic forms of haematoporphyrin(LX) dihydrochloride (HpdiCl) on solvent composition was investigated. In 2.8 x 10(-4) M solutions of HpdiCl in apolar (C6H6) and polar (CH3CN) solvents, HpdiCl exists in dicationic form. In hydrogen-bonding solvents, such as CH3OH, HpdiCl can exist in neutral, monocationic and dicationic forms. In C6H6-CH3OH solvent mixtures, the ionic forms in which HpdiCl is present depend on the composition of the solvent and on the acidity of the solution. The rate of oxidative photodegradation of HpdiCl excitation in its Q bands (WBI) and the ability to produce free radicals are different for the different ionic species. The highest values correspond to the dicationic form of HpdiCl and the lowest values correspond to the neutral species. In the absence of oxygen, the formation of free radicals due to the reaction of 3(Hp dication) is detected in the following solvent mixtures: CH3OH-toluene, CH3OH-ethylbenzene, CH3OH-hexane. The data obtained indicate that interaction of 3(Hp dication) with methine groups is an intermediate step in the formation of free radicals. In the HpdiCl concentration range studied, the presence of a phenolic antioxidant, such as beta-naphtol, inhibits the oxidative photodegradation of the dicationic form in a treated solution, but has little effect on the oxidative photobleaching of the monocation. The rate of oxidative photodegradation of the monocationic form increases with the addition of propionic acid to the solution.

A long-lived transient resulting from flash photolysis of hematoporphyrin in aqueous solution.

A long-lived transient with a lifetime of several hundred microseconds was observed following the flash photolysis of aqueous solutions of hematoporphyrin buffered at pH 7.5. The transient-ground state difference absorption spectrum was determined 500 microseconds after flash photolysis. The yield of this species was found to increase with increasing hematoporphyrin concentration and it was also found to depend on the excitation wavelength. The lifetime of the species is not significantly affected by the presence of oxygen. Because the triplet state of hematoporphyrin is not the only long-lived species produced by flash photolysis of aqueous hematoporphyrin solutions, the observed triplet state extinction coefficients will be lower than the true value and hence the triplet state yields of hematoporphyrin determined by the flash photolysis, "complete conversion" technique, are only upper limits. The formation of the long-lived species is discussed in terms of electron transfer between the monomer partners in hematoporphyrin dimer and aggregates which are present in aqueous solutions of hematoporphyrin, particularly in concentrated solutions.

The photophysics of haematoporphyrin dimers or aggregates in aqueous solution.

The triplet lifetime and decay rate law observed from haematoporphyrin in aqueous solution were found to depend on concentration with the triplet-state decay being very much slower in dilute solution than in more concentrated solutions. The wavelength of excitation also has an effect on the decay kinetics. In a 1 x 10(-4) mol dm-3 solution the decay resulting from flash photolysis at 527 nm is satisfactorily described by a single exponential having a decay constant of 1.5 x 10(4) S-1 and was ascribed to the triplet state of a haematoporphyrin dimer or aggregate. However, excitation of a haematoporphyrin solution of the same concentration at 351 nm results in a slower triplet-state decay which is indistinguishable, within experimental error, from that observed from a dilute haematoporphyrin solution excited at 527 nm and which is ascribed to the monomer triplet state. Although resolved Soret band absorption spectra indicate that in a 1 x 10(-4) mol dm-3 aqueous solution of haematoporphyrin most radiation at 351 nm is absorbed by a dimer, the decay following flash photolysis at 351 nm is dominated by the triplet state of the haematoporphyrin monomer. This implies that the excited singlet state of the dimer whose ground state absorbs the shorter-wavelength region of the Soret band undergoes efficient intersystem crossing to the monomer triplet state.(ABSTRACT TRUNCATED AT 250 WORDS)

Generation of free radicals by photoexcitation of pheophorbide alpha, haematoporphyrin and protoporphyrin.

An investigation of the production of radical species by photoexcitation pheophorbide alpha, haematoporphyrin and protoporphyrin was performed. In an aqueous solution containing different amounts of ethanol, the superoxide radical was detected by the spin trapping technique. In addition, secondary radicals were observed. The generation of oxygen radicals was found to dominate in solutions with a low ethanol content.

Variation in the fluorescence decay properties of haematoporphyrin derivative during its conversion to photoproducts.

Haematoporphyrin derivative (HpD) photoproducts are formed in aqueous solutions during light exposure in the presence of oxygen. The evaluation of the fluorescence decay of the photoproduct-enriched HpD solution shows an increase in the short-lived components, especially about 2 ns, in comparison with HpD without photoproducts. The bleaching of the HpD fluorescence and the photoproduct formation by the fluorescence-exciting radiation has to be taken into account in the evaluation of stationary as well as time-resolved fluorescence measurements.

Phototransformations of porphyrins in aqueous and micellar media.

Porphyrin photoproducts with absorption maxima at 640 and 660 nm are formed in aqueous and micellar solutions during light exposure. Changes in the dimethoxyhaematoporphyrin (DMHp) Soret band on irradiation suggest that the photoproduct (640 nm) formation is conditioned by the "sandwich"-type self-associates. The formation of the photoproduct (660 nm) in micellar haematoporphyrin (Hp) and photosan-3 (PS) solutions and its formation in small amounts in phosphate-buffered solution (PBS), is related to the presence of covalently linked porphyrin structures and/or interaction with surfactant molecules. PS is the most photostable of the three investigated porphyrins. Its photostability is probably due to the presence of covalently linked "sandwich"-type aggregates.