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.

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.

Total synthesis of hematoporphyrin and protoporphyrin; a conceptually new approach.

The total synthesis of protoporphyrin IX and its disodium salt using a new alternative method to the classical MacDonald condensation is reported. The key step is the reaction of the new unsymmetrical diiodo dipyrrylmethane 1 with the known dipyrrylmethane 2. Coupling of the two fragments leads directly to porphyrin 3 without the need of an oxidizing agent. The new methodology is well suited for the synthesis of protoporphyrin IX derivatives on a multi-100 g scale in good quality without the need for chromatography. Furthermore, these preparations are completely free of any contaminant of animal origin, which represents a real improvement in the manufacturing of protoporphyrin IX derivatives.

Synthesis, radiolabeling, biodistribution and fluorescent imaging of histidine-coupled hematoporphyrin.

INTRODUCTION: Hematoporphyrin (Hp) and hematoporphyrin derivatives (HpDs) have been widely used as photosensitizers in photodynamic therapy (PDT). Radiolabeling of HpDs is helpful for preclinical and clinical studies of PDT. METHODS: The histidine-coupled hematoporphyrin (His-Hp) was synthesized and radiolabeled with [(99m)Tc(CO)(3)(H(2)O)(3)](+). Biodistribution of the radioligand and fluorescent imaging of His-Hp in mice bearing S180 tumor were investigated. RESULTS: [(99m)Tc(CO)(3)](+)-labeled His-Hp was electrically neutral, hydrophilic and stable. The biodistribution of the radioligand in S180 tumor-bearing mice was similar with that of nonlabeled HpD in the literature. The uptake of His-Hp in tumors and livers was confirmed by fluorescent imaging. CONCLUSIONS: The complex [(99m)Tc(CO)(3)](+)-His-Hp might be suitable for in vivo dose evaluation of HpD in PDT.

New two-photon activated photodynamic therapy sensitizers induce xenograft tumor regressions after near-IR laser treatment through the body of the host mouse.

PURPOSE: The aim of this study was to show that novel photodynamic therapy (PDT) sensitizers can be activated by two-photon absorption in the near-IR region of the spectrum and to show, for the first time, that such activation can lead to tumor regressions at significant tissue depth. These experiments also evaluated effects of high-energy femtosecond pulsed laser irradiation on normal tissues and characterized the response of xenograft tumors to our PDT protocols. EXPERIMENTAL DESIGN: Human small cell lung cancer (NCI-H69), non-small cell lung cancer (A549), and breast cancer (MDA-MB-231) xenografts were induced in SCID mice. Irradiation of sensitized tumors was undertaken through the bodies of tumor-bearing mice to give a treatment depth of 2 cm. Posttreatment tumor regressions and histopathology were carried out to determine the nature of the response to these new PDT agents. Microarray expression profiles were conducted to assess the similarity of responses to single and two-photon activated PDT. RESULTS: Regressions of all tumor types tested were seen. Histopathology was consistent with known PDT effects, and no, or minimal, changes were noted in irradiated normal tissues. Cluster analysis of microarray expression profiling showed reproducible changes in transcripts associated with apoptosis, stress, oxygen transport, and gene regulation. CONCLUSIONS: These new PDT sensitizers can be used at a depth of 2 cm to produce excellent xenograft regressions. The tumor response was consistent with known responses to single-photon activated PDT. Experiments in larger animals are warranted to determine the maximal achievable depth of treatment.

Preparation and in vitro biological evaluation of tetrapyrrole ethanolamide derivatives as potential anticancer agents.

Tetrapyrrole ethanolamide derivatives, 1 and 2, were prepared from hematoporphyrin IX (HPIX, 3) and methyl pheophorbide a (mPheo, 6). These were evaluated for their dual action as chemotherapeutics and photosensitizers in treatment of cancer. The novel compounds showed significant in vitro anticancer activity as measured in different cell lines using the MTT assay and photodynamic activity measured by erythrocytes' photohemolysis.

Synthesis and evaluation of a hematoporphyrin derivative in a folate receptor-targeted solid-lipid nanoparticle formulation.

PURPOSE: This study was aimed at the synthesis, formulation and in vitro evaluation of folate receptor (FR)-targeted solid-lipid nanoparticles (SLNs) as a carrier for a lipophilic derivative of the photosensitizer hematoporphyrin (Hp), in FR-overexpressing tumor cells. MATERIALS AND METHODS: FR-targeted hematoporphyrin-stearylamine (HpSa) SLN composed of Triolein:Egg-phosphatidylcholine (EPC):Tween-80 (T-80) (64:25:10), with 0.5 mole % of folate-polyethyleneglycol-cholesterol (FPC) or polyethyleneglycol-distearoylphosphatidylethanolamine (PEG-DSPE), were prepared by ethanol injection method. Stability of the SLN was monitored by changes in particle size at 4 degrees C and drug retention at various time points. Cellular uptake and IC50 values of the FR-targeted formulations were determined in vitro in the FR (+) KB cells. RESULTS: Stable targeted SLNs were prepared by ethanol injection encapsulating greater than 95 percent of 5 mole % of HpSa, having a mean diameter < 200 nm. In vitro cytotoxicity assay on the FR-targeted SLN gave IC50 of 1.57 microM in KB cells and non-targeted SLNs gave an IC50 of 5.17 microM. FR selectivity was confirmed by fluorescence microscopy. CONCLUSION: FR-targeted SLNs incorporating the lipophilic drug HpSa were capable of specific receptor binding in cultured KB cells, which warrants further investigation.

Synthesis, biodistribution and antitumor activity of hematoporphyrin-platinum(II) conjugates.

A new series of platinum(II) complexes of pegylated hematoporphyrin derivatives with controlled hydrophobic/hydrophilic balance were synthesized by introducing different kinds of poly(ethylene glycol) and amine ligands to the porphyrin ring. The antitumor activity of the porphyrin-platinum(II) conjugates was assayed in vitro and in vivo against leukemia L1210 cell line and various human tumor cell lines. The present complexes exhibited high antitumor activity and improved water solubility as well as considerable lipophilicity. In particular, complex 16 showed not only higher in vivo activity (T/C%=258) than cisplatin (T/C%=184) and carboplatin (T/C%=168), but also excellent solubility in water and organic solvent. The antitumor activity of complex 20 was superior to that of carboplatin against all human tumor cell lines tested. Moreover, some amphiphilic complexes (7 and 12) exhibited elevated tumor-localizing effect (tumor/muscle ratio>2).

Hematoporphyrin-derived soluble porphyrin-platinum conjugates with combined cytotoxic and phototoxic antitumor activity.

To combine the cytotoxic activity of cisplatin and the phototoxicicity of hematoporphyrin derivatives in the same molecule, hematoporphyrin was derivatized at the two secondary alcohol positions by etherification with oligo- and poly(ethylene glycol) units. The two carboxylic acid groups of the propionate side chains were used to bind platinum fragments. The antiproliferative activity of 35 platinum complexes (0.5, 1, and 5 microM) differing in solubility and type of the platinum fragment and the corresponding porphyrin ligands were studied in tests with TCC-SUP and J82 transitional bladder cancer cells in the dark and after irradiation (lambda = 600-730 nm, 24 J/cm(2)). The most active compounds were found among the porphyrin-platinum conjugates bearing the diammine and (RR/SS)-trans-1,2-diaminocyclohexane ligand. These porphyrin-platinum conjugates, especially the water-soluble species, such as diammine(7,12-bis[1-(poly(ethylene glycol)-750-monomethyl ether-1-yl)ethyl]-3,8,13,17-tetramethylporphyrin-2,18-dipropionato)platinum(II), are promising candidates for the development of a novel type of photosensitizers with intrinsic cytotoxicity, which due to the porphyrin constituent may selectively enrich in tumor tissues.

An atomic basis of cancer (V): A new marker in animal tumors.

Particle-induced X-ray emission (PIXE) analysis can be applied to mark (or to localize) a cancer lesion in a benign environment in test animals. We synthesized hematoporphyrin derivative (HPD) with rare earth metals to form lanthanide-HPD to enhance the localizing efficiency. The sensitive PIXE detection of trace elements in amounts on the order of nanograms or less could possibly be used to probe carcinogenesis at the level of the cell. To explain the present approach, we present the results of an experiment in which we used paraseodynium-HPD localizer.

Photosensitizer targeting in photodynamic therapy. I. Conjugates of haematoporphyrin with albumin and transferrin.

Conjugates of haematoporphyrin (HP) with serum albumin and transferrin were prepared, purified by gel filtration and characterized by high performance liquid chromatography (HPLC), polyacrylamide gel electrophoresis (PAGE) and spectroscopy. Although the fluorescence was somewhat quenched, the conjugates had similar singlet oxygen quantum yields to free porphyrin. The albumin conjugate (HP-BSA) could be divided into monomeric and cross-linked fractions. In NIH 3T3 and HT29 cells, native albumin could not compete with the uptake of HP-BSA and the uptake was greatly enhanced in the absence of serum and in the presence of poly-L-lysine. We infer that the conjugate was mostly associated with the plasma membrane in these cells. The uptake of HP-transferrin showed evidence of a receptor-mediated component in that it was partially inhibited by native protein and increased when transferrin receptors were upregulated by an iron chelator. J774 macrophage-like cells accumulated fluorescence from HP-BSA to a much higher degree than HT29 cells, even though the protein was extensively degraded (HT29 cells did not appear to degrade the protein). The time course of the photocytotoxicity of HP-BSA was prolonged in J774 cells, although their response to free porphyrins was similar to that seen in HT29 cells. Chloroquine inhibited protein degradation without having an effect on the fluorescence uptake. J774 cells acquired more fluorescence and degraded more protein when supplied with cross-linked HP-BSA compared with monomeric fraction. For a given fluorescence uptake, the cross-linked fraction was also more photocytotoxic. We conclude that macrophages can acquire photosensitizer-protein conjugates avidly and that these are delivered to the lysosomes. These types of conjugate may have applications in targeting fluorescent molecules for diagnostic imaging and for the photodynamic treatment of macrophage malignancies.

Haematoporphyrin derivatives: distribution in a living organism.

Pharmacokinetics of accumulation in organs and tissues was studied for two haematoporphyrin-based photosensitizers. These sensitizers, haematoporphyrin derivative (HpD) and an oligomeric haematoporphyrin (OHp), contained different amounts of monomeric fraction (25% and 5% respectively) and in OHp the macrocycles were bonded together with ether bonds. OHp was shown to accumulate in tumours in higher amounts than HpD. The maximal tumour to tissue concentration ratio for OHp was 6.7 observed 54 h after injection; the same ratio for HpD was 2.8 after 48 h.

[Synthesis of porphyrin derivatives].

This paper presents the preparation of eleven 2, 4 or 6, 7-substituted porphyrine derivatives II-XII from I or diethyl ether of IV. The effects of these compounds, with irradiation, on DNA synthesis in L1210 cells were studied. The results showed that (1) all of these compounds were effective while (2) Va or Vb, VI and VII were more effective.

Mn (III) hematoporphyrin. A potential MR contrast agent.

Manganese (III) hematoporphyrin (MnHP), a new and stable complex, was prepared, and its toxicity and magnetic resonance (MR) imaging properties were evaluated. In tests of acute and subacute toxicity, no deaths resulted from bolus intravenous injections of 13 or 19 mumols/kg of MnHP, but there was a 33% mortality when the dose was 38 mumols/kg. Laboratory results were normal in the surviving rats. Ultraviolet- visible spectroscopy of the urine and serum of two rats injected 24 hours previously with 38 mumols/kg MnHP revealed no free HP, suggesting in vivo stability of MnHP. Finally, using a standardized imaging protocol, there was a mean increase of 37% in the liver-to-muscle intensity ratios in four rats injected 24 hours previously with 25 mumols/kg MnHP when compared to paired controls (P less than .005). In addition, obvious visual increase in the signal intensity of the liver on T1-weighted images was seen in animals tested with 13 and 19 mumols/kg of MnHP. The results suggest that further evaluation of MnHP as an MR contrast agent for the liver is warranted.

Studies on the photodynamic effect of haematoporphyrin derivative.

A case-control photodynamic therapy (PDT) was studied on adenocarcinoma (AC755) in BDF1 mice. Haematoporphyrin derivative (HPD; Porphyrin Products, U.S.A.) and a Bulgarian HPD were used as photosensitizers at doses of 10 mg kg-1. An argon dye laser system with lambda em=630 nm (400 mW cm-2) was used for PDT with a total light dose of 400 J cm-2. The therapeutic effect was assessed by the changes in tumour dimensions, the size of photonecrosis and the mean survival time of the animals. Histologic and ultrastructural studies were carried out. No significant difference was recorded between the antitumour effects of the two photosensitizers. Best results were obtained in small tumours (less than 10 mm) with incision of covering skin. Results are discussed in an attempt to advocate an optimal regimen for PDT in experimental tumours.

Syntheses and photosensitizing activity of porphyrins joined with ester linkages.

In order to investigate the structure of hematoporphyrin derivative and its purified version, Photofrin-II, porphyrin dimers with ester linkage were synthesized. 2,4-Diacetyldeuteroporphyrin dimethyl ester and protoporphyrin IX dimethyl ester were used as starting materials. The methyl esters were replaced by trimethylsilylethyl esters to protect the carboxylic groups. Deprotection using tetra-n-butylammonium fluoride in tetrahydrofuran regenerated the carboxylic functions. Reversed phase high performance liquid chromatography was used to compare the synthetic dimers with components of Photofrin-II. Our data indicate that these dimers are not components of Photofrin-II. During the synthesis of a 13C-labeled dimer with an ester linkage, a small amount of trimer was also isolated. The structures of these compounds were confirmed by nuclear magnetic resonance and mass spectroscopy. Using a standard screening system with DBA/2 mice bearing transplanted SMT-F tumors, these dimers were found not to be as active as Photofrin-II.

Preparation and photosensitizing properties of hematoporphyrin ethers.

Hematoporphyrin ethers having acyl or aryl substituents in the 2 and 4 positions of the porphyrin ring have been synthesized, starting from protoporphyrin HBr adduct, and tested for photosensitizing efficiency on cells in vitro and transplanted tumors in mice. In general, they resemble the tumor localizing fraction of hematoporphyrin derivative (Hpd). Cellular uptake and retention runs parallel with the degree of their non-polarity and in vitro sensitizing efficiencies are up to ten times that of Hpd or Photofrin II (P II). They have high quantum yields for inactivation of cells and also relatively low in vivo skin/tumor concentration ratios.

On the preparation of dihematoporphyrin ether-free hematoporphyrin derivative.

A dihematoporphyrin ether-free hematoporphyrin derivative has been prepared by a base-catalysed dehydration of hematoporphyrin with sodium hydroxide. The identification was performed by HPLC and mass spectroscopy (FD-MS). The reaction of hematoporphyrin with 1 M sodium hydroxide for 24 h yields more than 90% of the monomeric porphyrins.