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A tetracationic meso-substituted amphiphilic porphyrin (abbreviated as C14) was encapsulated within silica microparticles to yield a conjugate with a mean particle diameter of ca. 0.9 μm. The conjugate displayed a complete stability for at least 3 months when suspended in a neutral aqueous medium. The encapsulated C14 underwent a limited photobleaching when the conjugate was exposed to full spectrum visible light. Illumination of the silica microparticle-bound C14 by visible light resulted in the generation of singlet oxygen and induced a decrease in the survival of 4 log for a 20 min irradiation of the Gram-positive bacterium meticillin-resistant Staphylococcus aureus (MRSA) and a 30 min irradiation of the Gram-negative bacterium Escherichia coli (E. coli). Under identical experimental conditions photoexcited free C14 caused a decrease in viability of 5 log for MRSA and 6 log for E. coli. When the conjugate loaded with 12 μM C14 was added to a water sample contaminated with MRSA (10(8) cells per ml) a tight association of the bacterial cells with the silica microparticle-porphyrin system was achieved. Subsequent illumination of the conjugate with visible light (30 min, 100 mW cm(-2)) caused a 3 log reduction in the population of MRSA cells in the water sample. Importantly, the conjugate was readily recovered by filtration of the aqueous suspension and shown to maintain a high antibacterial photoactivity when introduced into a new MRSA-contaminated medium and irradiated.

The increasing use of photosensitized processes for disinfection of microbiologically polluted waters requires a precise definition of the factors controlling the degree of photosensitivity in target and non-target organisms. In this regard, tests with protozoa and invertebrates which have a natural habitat in such waters may be used as first screening methods for the assessment of possible hazards for the ecosystem. A new cationic porphyrin, namely meso-tri(N-methyl-pyridyl)mono(N-dodecyl-pyridyl)porphine (C12), is tested in this work on the protozoan Ciliophora Colpoda inflata and Tetrahymena thermophila and the Crustacea Branchiopoda Artemia franciscana and Daphnia magna. The protocol involved 1 h incubation with porphyrin doses in the 0.1-10.0 μM range and subsequent irradiation with visible light at a fluence rate of 10 mW cm(-2). The results indicate that C12 porphyrin has a significant affinity for C. inflata and T. thermophila; this is also shown by fluorescence microscopic analyses. C. inflata cysts were resistant to the phototreatment up to a porphyrin dose of 0.6 μM. The effects of C12 on cysts have been evaluated at 3 and 24 h after the end of the phototreatment; a delay in the excystment process was observed. T. thermophila was fairly resistant to the phototreatment with C12 porphyrin. The data obtained with the two crustaceans indicated that the effects of dark- and photo-treatment with C12 need to be closely examined for every organism. A. franciscana is more resistant, probably owing to its ability to adapt to extreme conditions, while the high level of photosensitivity displayed by Daphnia magna represents a potential drawback, as this organism is often selected as a reference standard for assessing the environmental safety. Thus, while C12 photosensitisation can represent a useful tool for inducing a microbicidal or larvicidal action on polluted waters, the irradiation protocols must be carefully tailored to the nature of the specific water basin, and in particular to its biotic characteristics.

Biolarvicides, such as microbial formulations based on Bacillus thuringiensis and B. sphaericus, have been found to be highly effective against mosquito larvae and are currently employed as eco-friendly alternatives to synthetic chemical insecticides for vector control. Recently, a porphyrin of natural origin has been suggested as a sunlight-activatable larvicide against the dengue vector Aedes aegypti. In order to validate the approach for the control of the malaria vector, we tested the photo-larvicidal activity of a novel porphyrin, namely meso-tri(N-methyl-pyridyl), mono(N-dodecyl-pyridyl)porphine, C12, associated with two specifically selected carriers, against Anopheles gambiae s.s. and An. arabiensis larvae, both laboratory reared and collected from malaria endemic sites in Burkina Faso. Both C12-porphyrin formulates, when administered to larvae at a 50 μM porphyrin dose, were accumulated in the alimentary canal. Subsequent exposure of the porphyrin-loaded larvae to sunlight for short times (0.5–3 h) led to a complete mortality. The high efficacy exhibited by a “foodstuff” porphyrin formulate also in the presence of typical larval food particles opens promising perspectives for the development of an effective photocidal larvicide.

A water-soluble [meso-tetra(4-nido-carboranylphenyl)porphyrin] (H(2)TCP) bearing 36 boron atoms was studied for its accumulation and its radio/photo-sensitization efficiency towards murine melanotic melanoma cells. The amount of H(2)TCP in the cells increased with the porphyrin dose in the incubation medium up to 100 microM with no significant dark toxicity. Fluorescence microscopy observations showed that the porphyrin was largely localized intracellularly. Based on these "in vitro" results our investigations were pursued using the B16F1 melanotic melanoma subcutaneously transplanted in C57BL6 mice as "in vivo" model. Phormacokinetic studies were performed by injection of H(2)TCP intratumorally (1 mg/kg) and intravenously (10 mg/kg). At 0.5h after i.t. administration or at 24 h after i.v. injection, the amounts of (10)B in the tumour were about 60 ppm and about 6 ppm, respectively. The distribution of H(2)TCP in the tumour after intravenous or intratumoural injection was also assessed by fluorescence microscopy analyses. Under these conditions, preliminary BNCT studies were carried out using a new thermal column called HYTOR (HYbrid Thermal spectrum sHifter TapirO Reactor) inserted in the fast nuclear reactor Tapiro at Enea Casaccia, Italy. The mice were exposed to HYTHOR radiation field for 20 min at a reactor power of 5 kW. In spite of different amounts of (10)B in the tumour at the irradiation time, a similar significant delay in tumour growth (5-6 days) was induced by neutron irradiation in intratoumorally and intravenously injected mice. The response of the melanotic melanoma to H(2)TCP-BNCT was compared with that obtained by irradiation after intraperitoneal injection of boron-phenylalanine.

Background and Purpose: Boron Neutron Capture Therapy (BNCT) is a binary cancer treatment that exploits the short range particles released from a nuclear fission reaction involving the non-radioactive 10 B nucleus and low-energy neutrons for the destruction of tumor cells. In this perspective, porphyrins and phthalocyanines can represent a vehicle for the transport of significant amounts of boron to the neoplastic lesion. Material and Methods: B16F1 melanotic melanoma subcutaneously transplanted in C57/BL6 mice has been used as an in vivo model. Pharmacokinetic studies were performed by intratumoral and intravenous injection of a meso-substituted porphyrin containing 36 B atoms per molecule ( H 2 TCP ) and the distribution of H 2 TCP in the tumor was assessed by fluorescence microscopy analysis. The tumor-bearing mice were exposed to the radiation field for 20 min at a reactor power of 5 kW. Results: At 0.5 h after intratumoral administration or at 24 h after intravenous injection, the amount of 10 B in the tumor was found to be about 60 ppm and about 6 ppm, respectively. In spite of the different amounts of 10 B in the tumor at the time of irradiation, a very similar delay in tumor growth (5-6 days) was induced by neutron irradiation in the two groups of injected mice with respect to control mice. Conclusions: Our results demonstrate that a suitable boron-loaded porphyrin displays a significant affinity for subcutaneous tumors, and upon activation by thermal neutrons, can promote an important response even in a fairly aggressive and generally radioresistant tumor such as melanotic melanoma.

HYTHOR (HYbrid Thermal spectrum sHifter tapirO Reactor) is a new thermal-neutron irradiation facility, which was installed and became operative in mid 2005 at the TAPIRO (TAratura PIla Rapida potenza 0) fast reactor, in the Casaccia research centre (near Rome) of ENEA (Ente per le Nuove tecnologie Energia ed Ambiente). The facility has been designed for in vivo radiobiological studies. In HYTHOR irradiation cavity, 1–6 mice can be simultaneously irradiated to study skin melanoma treatments with the BNCT (boron neutron capture therapy). The therapeutic effects of HYTHOR radiation field on mouse melanoma has been studied as a preliminary investigation before studying the tumour local control due to boron neutron capture effect after boronated molecule injection. The method to properly irradiate small animals has been precisely defined. Results show that HYTHOR radiation field is by itself effective in reducing the tumour-growth rate. This finding has to be taken into account in studying the effectiveness of new 10 B carriers. A method to properly measure the reduction of the tumour-growth rate is reported and discussed.

An amphiphilic tetracationic derivative of Zn(II)-phthalocyanine (RLP068) was prepared by means of chemical synthesis and was showed to possess efficient photophysical and photosensitizing properties against model biological substrates. RLP068 was incorporated into a gel formulation, which allowed its ready penetration into the epidermal layers, but not into the dermis, of both Balb/c and hairless SKH1 mice after 1-2 h of topical deposition. Pharmacokinetic studies showed that the phthalocyanine thus formulated does not enter the general blood circulation. The epidermis-associated amount of phthalocyanine was sufficient to cause an important cutaneous damage upon irradiation with red light (600-700 nm; 100-180 mW/cm 2 , 160 J/ cm 2 ); the latter was confined to the epidermal area with no apparent diffusion to the underlying dermal layers or appearance of photosensitivity in distal skin areas. A systematic investigation of the interplay among the different parameters (deposition time of the formulated phthalocyanine on mouse skin, irradiation fluence rate and total light fluence) allowed us to identify the minimal phototoxic dose, as well as to define irradiation protocols allowing the repeatability of the phototherapeutic treatment. The potential of RLP068 to act as a PDT agent for cutaneous diseases is briefly discussed.

Photodynamic therapy with full spectrum visible light and porphyrin-type photosensitisers appears to efficiently induce the inactivation of a broad spectrum of microbial pathogens, including antibiotic-resistant strains, without in turn promoting the selection of PDT-resistant species. Application of this therapeutic modality to the treatment of spontaneously developed infections in dogs resulted in a very efficient healing of wounds with an extensive drop in the population of pathogens. The treatment appeared to be applicable for both mycotic and bacterial infections and was devoid of detectable undesired side effects. No significant differences in efficacy was observed whether a porphyrin, chlorin, or phthalocyanine photosensitising agent was used in spite of strong differences in the light absorption spectrum among the three tetrapyrrole derivatives.

Incubation of B78H1 amelanotic melanoma cells with a potential photothermal sensitizer, namely, liposome-incorporated Ni(II)-octabutoxy-naphthalocyanine (NiNc), induces an appreciable cellular accumulation of the naphthalocyanine, which is dependent on both the NiNc concentration and the incubation time. No detectable decrease in cell survival occurs upon red-light irradiation (corresponding to the longest-wavelength absorption bands of NiNc) in a continuous-wave (c.w.) regime of the naphthalocyanine-loaded cells. On the other hand, 850 nm irradiation with a Q-switched Ti:sapphire laser operating in a pulsed mode (30 ns pulses, 10 Hz, 200 mJ/pulse) induces an efficient cell death. Thus, ca. 98% decrease in cell survival is obtained upon 5 min irradiation of cells that have been incubated for 48 h with 5.1 μM NiNc. The efficiency of the photoprocess is strongly influenced by the NiNc cell incubation time prior to irradiation. Photothermal sensitization with NiNc appears to open new perspectives for therapeutic applications, as suggested by preliminary in vivo studies with C57/BL6 mice bearing a subcutaneously implanted amelanotic melanoma.

Three Si(IV)-tetradibenzobarreleno-octabutoxyphthalocyanines (TDiBOPcs) bearing different axial ligands on the metal ion were studied for their tumour-localizing and-photosensitizing properties after i.v. injection via a Cremophor emulsion (0.35 μmol kg −1 b.w.) to Balb/c mice bearing an intramuscularly implanted MS-2 fibrosarcoma. In all cases, the maximum tumour accumulation of the photosensitizer (0.8-1.9 nmol g −1 of tissue) was found at 24 h after injection. The efficiency and selectivity of tumour targeting appeared to be dependent on the nature of the axial ligands; optimal values of these parameters were obtained in the case of the bis (trihexyl-siloxy)-substituted Si(IV)-TDiBOPc, which gave a 7–9 tumour/muscle ratio of phthalocyanine concentration at 24–48 h after injection. The extent of tumour response to PDT treatment was correlated with the concentration of the photosensitizer in the tumour tissue: upon 740 nm irradiation (180 mW cm −2 , 200 J cm −2 ) at 48 h after injection of 0.35 μmol kg 1 of Si(IV)-TDiBOPcC 6 H 13 , the tumour growth exhibited a delay of about 7 days.

Introduction: Despite continuous advances in our knowledge of the biology of acute myelogenous leukemia (AML), the prognosis of AML patients treated with standard chemotherapy is still poor, especially in the elderly. Therefore, there is a need for novel targeted and less toxic therapies, particularly for patients who develop resistance to traditional chemotherapeutic drugs. Constitutively active phosphatidylinositol 3-kinase (PI3K) signaling characterizes many types of tumors, including AML, where it negatively influences response to therapeutic treatments. Areas covered: The literature data showed that small inhibitor molecules targeting PI3K signaling induced cell cycle arrest, apoptosis and decreased drug-resistance in AML cells. PI3K inhibitors were also capable of targeting leukemic initiating cells (LICs), the most relevant target for leukemia eradication, whereas they tended to spare healthy hematopoietic stem cells. Expert opinion: Data emerging from pre-clinical settings suggest that the PI3K pathway is critically involved in regulating proliferation, survival and drug-resistance of AML cells. Therefore, we propose that novel drugs targeting this signaling pathway may offer a novel and less toxic treatment option for AML patients, most likely in combination with a lower dosage of traditional chemotherapeutic agents or other innovative therapeutic agents.

Previous studies (Biolo et al., Photochem. Photobiol. 59, 362-365, 1994) showed that liposome-delivered Si(IV)-naphthalocyanine (SiNc) photosensitizes B16 pigmented melanoma subcutaneously transplanted in C57 mice to the action of 776 nm light. However, the efficacy of the phototreatment was limited by a lack of selectivity of tumor targeting by SiNc as well as by incomplete necrosis of the neoplastic mass. The present investigations show that the use of a different delivery system (Cremophor emulsion vs liposomes of dipalmitoylphosphatidylcholine) causes no significant increase in the selectivity of tumor targeting for three injected doses of SiNc (0.5, 1, 2 mg/kg). However, upon 776 nm light irradiation (300 mW/cm2; 520 J/cm2), the delay in the rate of tumor growth was maximal (7-8 days) for the highest naphthalocyanine dose. On the other hand, a remarkable improvement in the tumor response was obtained by inducing an intratumoral temperature increase to 44 degrees C immediately after PDT. The thermal effect appeared to be due to photoexcitation of melanin by 776 nm light (550 mW/cm2; 520 J/cm2) and subsequent partial conversion of absorbed energy into heat.

Four Ge(IV)-octabutoxy-phthalocyanines (GePcs) bearing two alkyl-type axial ligands were assayed for their pharmacokinetic properties and phototherapeutic efficiency in Balb/c mice bearing an intramuscularly transplanted MS-2 fibrosarcoma. The GePcs were i.v. injected at a dose of 0.35 mumol kg-1 body weight after incorporation into either Cremophor emulsions or small unilamellar liposomes of dipalmitoyl-phosphatidylcholine (DPPC). Both the nature of the delivery system and the chemical structure of the phthalocyanine were found to affect the behaviour of the GePcs in vivo. Thus, Cremophor-administered GePcs invariably yielded a more prolonged serum retention and a larger association with low-density lipoproteins (LDLs) as compared with the corresponding liposome-delivered phthalocyanines. This led to a greater efficiency and selectivity of tumour targeting. These effects were more pronounced for those GePcs having relatively long alkyl chains (hexyl to decyl) in the axial ligands. Maximal tumour accumulation (0.67 nmol per g of tissue) was found for Ge-Pc(hexyl)2 at 24 h after injection. Consistently, the Ge-Pc(hexyl)2, administered via Cremophor, showed the highest phototherapeutic activity towards MS-2 fibrosarcoma.

Porphyrins have been shown to act as very efficient photosensitizing agents against a broad number of microbial pathogens, including bacteria, fungi, and protozoa. This property has promising applications at a clinical level for the treatment of infectious diseases by photodynamic therapy. Moreover, this technique is also being used to address environmental problems of high significance, such as the decontamination of wastewaters, the disinfection of fish-farming tanks, the protection of animal species (e.g., amphibians and reptiles) that are endangered by pathogens whose life cycle takes place largely in aqueous media, and the control of populations of noxious insects. Such diversified applications take advantage of the availability of a truly large number of porphyrin derivatives with chemical structures that can be tailored to comply with the physical and chemical properties as well as the biological features of several milieus. In addition, the property typical of porphyrins to absorb essentially all of the wavelengths in the sun emission spectrum allows the promotion of processes largely based on natural resources with significant energy savings and low impact on ecosystems.

PDT of infectious diseases of microbial origin is based on the property of selected photosensitising agents, especially those characterized by the presence of positively charged functional groups, to readily bind with the outer wall of bacterial, fungal and protozoan cells; once activated by irradiation with visible light, the photosensitisers generate reactive oxygen species which induce the irreversible modification of specific constituents of the wall. The consequent enhanced permeability of the wall allows sufficiently large doses of the photosensitiser to penetrate to the cytoplasmic membrane. The photoprocess then involves a number of endocellular activities (e.g., enzymic catalysis, metabolic pathways) whose integrity is critical for cell survival. The genetic material is generally involved in the late stage of the photoprocess, which minimizes the probability of inducing mutagenic processes. A suitable choice of the irradiation protocol usually leads to an extensive killing of microbial pathogens with no or minimal damage to host tissues. PDT differs from other usually adopted therapeutic modalities for the broad spectrum of action, the efficacy against antibiotic resistant cells, and the lack of selection of photoresistant strains.

The efficiency of a Zn(II)-phthalocyanine disulphide (C11Pc), a compound with both phthalocyanine units bearing seven hexyl chains and a sulphur terminated C11 chain, as a photodynamic therapy (PDT) agent was investigated in C57 mice bearing a sub-cutaneously transplanted amelanotic melanoma. The phthalocyanine was intravenously injected at a dose of 1.5 micromol/kg body weight either free or bound to gold nanoparticles, using a Cremophor emulsion as a delivery vehicle. Biodistribution studies at selected post-injection times showed that the nanoparticle-associated C11Pc was recovered in significantly larger amounts from all the examined tissues and the serum and yielded a greater selectivity of tumour targeting: thus, the ratio between the amount of phthalocyanine recovered from the amelanotic melanoma and the skin (peritumoural tissue) increased from 2.3 to 5.5 from the free to the gold nanoparticle-bound C11Pc at 24 h after injection. PDT studies with the C11Pc-loaded amelanotic melanoma showed a markedly more significant response of the tumour in the mice that had received the nanoparticle-bound photosensitiser; the PDT effect was especially extensive if the irradiation was performed at 3h after C11Pc injection when large phthalocyanine amounts were still present in the serum. This suggests that the PDT promoted by C11Pc predominantly acts via vascular damage at least in this specific animal model. This hypothesis was fully confirmed by electron microscopy observations of tumour specimens obtained at different times after the end of PDT, showing an extensive damage of the blood capillaries and the endothelial cells.

We describe the total synthesis and biological properties of a new carboranyl-containing chlorin (TPFC) that might find application as a dual sensitizer in the PDT and BNCT treatment of cancer. TPFC was found to be non-toxic in the dark but showed extensive photosensitizing ability both in vitro and in vivo despite its relatively low singlet oxygen quantum yield. In particular, TPFC exhibited significant photosensitizing activity against highly pigmented melanotic melanoma tumors in mice.

A water-soluble meso-substituted porphyrin (H(2)TCP) bearing 36 boron atoms, which appeared to be an efficient photodynamic sensitiser (singlet oxygen quantum yield=0.44), was studied for its accumulation by murine melanotic melanoma cells (B16F1). The amount of H(2)TCP in the cells increased with the porphyrin dose in the incubation medium up to, and at least, 100 microM concentrations with no significant cytotoxic effect in the dark. Moreover, the H(2)TCP uptake increased with the incubation time reaching a plateau value corresponding with the recovery of 0.4 nmol of H(2)TCP per mg of cell proteins after 24h incubation. Fluorescence microscopy observations showed that the porphyrin was largely localized intracellularly, exhibiting a discrete distribution in the cytoplasm with a pattern which was closely similar to that observed for the endosomal probe Lucifer yellow. The photosensitising efficiency of the H(2)TCP toward B16F1 cells was studied for different irradiation (1-15 min) and incubation (1-24 h) times. Nearly complete (95%) cell mortality was obtained upon incubation with 20 microM H(2)TCP and 10 min irradiation with red light (600-700 nm, 20 mW/cm(2)). The porphyrin was also accumulated in appreciable amounts by the tumour tissue after intravenous injection to C57BL/6 mice bearing a subcutaneously transplanted melanotic melanoma. Maximum accumulation in the tumour was achieved by administration of H(2)TCP dissolved in the ternary mixture 20% dimethylsulfoxide (DMSO)-30% polyethyleneglycol (PEG 400)-50% water. Thus, this porphyrin could act as both a photodynamic therapy agent and a radiosensitising agent for boron neutron capture therapy.

Background and Objectives: Photodynamic therapy (PDT) appears to be endowed with several favorable features for the treatment of infections originated by microbial pathogens, including a broad spectrum of action, the efficient inactivation of antibiotic-resistant strains, the low mutagenic potential, and the lack of selection of photoresistant microbial cells. Therefore, intensive studies are being pursued in order to define the scope and field of application of this approach. Results: Optimal cytocidal activity against a large variety of bacterial, fungal, and protozoan pathogens has been found to be typical of photosensitizers that are positively charged at physiological pH values (e.g., for the presence of quaternarized amino groups or the association with polylysine moieties) and are characterized by a moderate hydrophobicity (n-octanol/water partition coefficient around 10). These photosensitizers in a micromolar concentration can induce a > 4–5 log decrease in the microbial population after incubation times as short as 5– 10 minutes and irradiation under mild experimental conditions, such as fluence-rates around 50 mW/cm 2 and irradiation times shorter than 15 minutes. Conclusions: PDT appears to represent an efficacious alternative modality for the treatment of localized microbial infections through the in situ application of the photosensitizer followed by irradiation of the photosensitizer-loaded infected area. Proposed clinical fields of interest of antimicrobial PDT include the treatment of chronic ulcers, infected burns, acne vulgaris, and a variety of oral infections. Lasers Surg. Med. 38:468–481, 2006. 2006 Wiley-Liss, Inc.

Two tetrasubstituted (RLP024 and RLP040) and one monosubstituted (MRLP101) Zn-phthalocyanines were readily accumulated by three skin-derived cell lines (HT-1080 transformed human fibroblasts, 3T3 mouse embryo fibroblasts and HaCaT human keratinocytes) upon 1 h-incubation with 0.5–5 μM phthalocyanine concentrations. The affinity was markedly larger for the tetra- as compared with the mono-substituted phthalocyanine, even though smaller phthalocyanine amounts were generally recovered from keratinocytes. As a consequence, the two tetra-substituted phthalocyanines exhibited a higher phototoxicity against all the three cell lines. Typically, the cell survival decreased by at least 80% after 1 min irradiation with 600–700 nm light at a fluence-rate of 50 mW/cm 2 in the presence of 5 μM phthalocyanine. Fluorescence microscopy and caspase-3 activation studies indicate that cell death of fibroblasts largely occurred by a random-necrotic process while the keratinocytes underwent cell death predominantly via apoptosis in spite of a very similar pattern of subcellular distribution of the phthalocyanines.

The synthesis of a Zn(ii)-phthalocyanine derivative bearing four 10B-enriched o-carboranyl units (10B-ZnB4Pc) and its natural isotopic abundance analogue (ZnB4Pc) in the peripheral positions of the tetraazaisoindole macrocycle is presented. The photophysical properties of ZnB4Pc, as tested against model biological systems, were found to be similar with those typical of other photodynamically active porphyrin-type photosensitisers, including a singlet oxygen quantum yield of 0.67. The carboranyl-carrying phthalocyanine was efficiently accumulated by B16F1 melanotic melanoma cells in vitro, appeared to be partitioned in at least some subcellular organelles and, upon red light irradiation, induced extensive cell mortality. Moreover, ZnB4Pc, once i.v.-injected to C57BL/6 mice bearing a subcutaneously transplanted pigmented melanoma, photosensitised an important tumour response, provided that the irradiation at 600-700 nm was performed 3 h after the phthalocyanine administration, when appreciable concentrations of ZnB4Pc were still present in the serum. Analogously, irradiation of the 10B-ZnB4Pc-loaded pigmented melanoma with thermal neutrons 24 h after injection led to a 4 day delay in tumour growth as compared with control untreated mice. These results open the possibility to use one chemical compound as both a photosensitising and a radiosensitising agent for the treatment of tumours by the combined application of photodynamic therapy and boron neutron capture therapy.

Studies on the synthesis, structural elucidation, and biological evaluation of new conjugates of poly-S-lysine with meso-substituted porphyrins are described. The new conjugates were used in the photoinactivation of antibiotic-resistant Gram-positive bacteria (Staphylococcus aureus strains ATCC 25923 and MRSA 110) and Gram-negative bacteria (Escherichia coli strain O4). The results show that the cationic conjugates are able to photosensitize the efficient inactivation of both types of bacteria.

An important factor in determining the efficacy of photosensitizing compounds in photodynamic therapy of tumors is the level to which tumors take up the photosensitizers after systemic injection. This parameter seems to be related to the transport modalities of the photosensitizer in the bloodstream. In this work the photosensitizer Zn(II)-tetradibenzobarrelenooctabutoxyphthalocyanine was shown to have an unprecedentedly high association with low-density lipoproteins (71% of the phthalocyanine in the plasma) when delivered in Cremophor micelles to tumor-bearing mice. This was accompanied by a particularly high tumor uptake at 24 h post-injection.

A number of Zn(II)- phthalocyanines bearing peripheral substituents of cationic nature due to the presence of quaternarized anilinium or ammonium groups were shown to be efficient photoantimicrobial agents: a 4-5 log decrease in the survival of both wild-type or methicillin-resistant Staphylococcus aureus was obtained upon short irradiation times in the presence of phthalocyanine concentrations as low as 0.1 microM. A careful selection of the experimental protocol, and in particular the use of short (5 min) incubation times and mild irradiation parameters, allowed one to achieve a high selectivity of S. aureus photoinactivation as compared with important constituents of potential host tissues, such as human fibroblasts and keratinocytes. The efficiency and selectivity of the photoprocess were not affected by the presence of 5% human serum.

Cu(II)-hematoporphyrin (CuHp) was efficiently accumulated by B78H1 amelanotic melanoma cells upon incubation with porphyrin concentrations up to 52 microM. When the cells incubated for 18 h with 13 microM CuHp were irradiated with 532 nm light from a Q-switched Nd: YAG laser operated in a pulsed mode (10 ns pulses, 10 Hz) a significant decrease in cell survival was observed. The cell photoinactivation was not the consequence of a photodynamic process, as CuHp gave no detectable triplet signal upon laser flash photolysis excitation and no decrease in cell survival was observed upon continuous wave irradiation. Thus, it is likely that CuHp sensitization takes place by photothermal pathways. The efficiency of the photoprocess was modulated by different parameters; thus, while varying the amount of added CuHp in the 3.25-26 microM range had little effect, pulse energies larger than 50 mJ and irradiation times of at least 120 s were necessary to induce a cell inactivation of about 50%. The porphyrin-cell incubation time prior to irradiation had a major influence on cell survival, suggesting that the nature of the CuHp microenvironment can control the efficiency of photothermal sensitization.

Benzoporphyrin derivative monoacid ring A (verteporfin, BPD-MA) when intravenously injected (5.5 μmol kg−1) to C57/BL6 mice bearing a subcutaneously transplanted B1 melanoma gave a maximal accumulation in the tumour within 1–3 h with recoveries of 1.84– 1.96 μmol kg−1. Irradiation of BPD-MA-loaded melanoma with 690-nm light from a dye laser at 3 h and 9 h post injection induced tumour necrosis and delay of tumour growth of 28 and 14 days respectively. The response of the tumour to BPD-MA photosensitization was enhanced by pretreatment with 1064-nm light from a pulse-operated Nd:YAG laser, which caused a selective breakdown of melanosomes. © 1999 Cancer Research Campaign

Irradiation of B16 pigmented melanoma subcutaneously transplanted in C57 mice with a single 650 mj pulse (10 ns) of 1064 nm light from a Q-switched Nd: YAG laser caused instantaneous bleaching of the pigmented tissue. Visual and histological examination of the resulting gray-colored tumor revealed the breakdown of melanosomes with no detectable alteration of the normal and tumor-overlying skin. Histological examination of the irradiated tumor showed some degree of vascular damage; the depth of the photodamage was not affected by the successive delivery of three consecutive light pulses. The bleached tumor grew at a modestly slower rate but the high-peak-power (HPP) laser treatment did not affect the tumor concentration of a photodynamic sensitizer Si(IV)-naphthalocyanine (isoBO-SiNc) intravenously injected 24 h before Nd : YAG irradiation. Treatment of the B16 pigmented melanoma by photodynamic therapy (PDT: 1 mg/kg isoBO-SiNc, 300 mW/cm2, 520 J/cm2) from a 774 nm diode laser immediately after the 1064 nm irradiation resulted in a 16 day delay of tumor regrowth, which was markedly longer than the delay (ca 6 days) obtained after PDT under identical conditions without the preirradia-tion. Thus, pretreatment of pigmented tumors with HPP 1064 nm light appears to enhance their susceptibility to conventional PDT. The tumor response was further enhanced by repeating the combined HPP/PDT treatment at an interval of 10 days (regrowth delay: 27 days), as well as by applying hyperthermia immediately after HPP/PDT (regrowth delay: ca 34 days).

A family of benzoporphyrins formed by differently substituted metallo tetrabenzoporphyrins and one opp-dibenzoporphyrin has been prepared. The former benzoporphyrins, and meso- tetra(m-hydroxy-phenyl)chlorin (m-THPC) to act as reference, have been encapsulated into liposomes and subjected to preliminary in vitro and in vivo assays to test their efficacy as photosensitizers in the photodynamic therapy of cancer. The results of the photocytotoxicity test shows that, with the exception of the nickel complexes 5, and 7/8, the other porphyrins are photobiologically active, the Mg-tetrabenzoporphyrin 1 and the opp-dibenzoporphyrin 10 being the most active. The dark toxicity of the photoactive porphyrins is in the range described for many photosensitizers, including HPD. The in vivo assays show no toxicity in the liver for any of the compounds tested, and also that 2 is the most promising photosensitizer among them, because of an efficient localization in an experimental mouse tumor.© (1996) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Melanotic malignant melanoma represents a major cause of death among skin neoplasias and its incidence is steadily increasing in Western world probably owing to changes in lifestyle which favour brief but intense exposures to sunlight, as well as due to enhanced levels of UV-B reaching the earth surface (1,2). White surgery is still the elective treatment for melanoma, alternative methods, including radiotherapy or immunotherapy, give a generally poor outcome (3).

The pharmacokinetic properties of the Ge(IV)-octabutoxy-phthalocyanines (GePc) with two axially ligated triethyl-siloxy (GePcEt) or trihexyl-siloxy (GePcHex) chains were studied in BALB/C mice bearing a transplanted MS-2 fibrosarcoma. The GePcs were delivered to mice after incorporation into unilamellar liposomes of dipalmitoyl phosphatidylcholine (DPPC) or in an emulsion of Cremophor-EL. The Cremophor delivered GePcs were cleared from the blood circulation at a much slower rate than the liposome-delivered GePcs. At the same time, Cremophor induced a slower and reduced uptake of the GePcs in the liver and spleen while it greatly enhanced the uptake in the tumour as compared to liposomes. Maximum tumour uptake was observed at 24 h post-injection and was equivalent to 0.67 and 0.50 nmol/g, respectively, for the Cremophor delivered GePcHex and GePcEt. The corresponding values for the liposome-delivered drugs were approximately one fourth of that observed with Cremophor.

The possibility of extending photodynamic therapy to the treatment of highly pigmented neoplastic lesions was tested by using Si(IV)-naphthalocyanine (SiNc) as a tumor-localizing agent. Si(IV)-naphthalocyanine displays intense absorbance at 776 nm (epsilon = 5 x 10(5) M-1 cm-1), where melanin absorption becomes weaker. As an experimental model we selected B16 pigmented melanoma subcutaneously transplanted to C57BL mice. Upon injection of 0.5 or 1 mg kg-1 of liposome-incorporated SiNc, maximal accumulation of the photosensitizer in the tumor was observed at 24 h with recoveries of 0.35 and 0.57 microgram g-1, respectively. However, the tumor targeting by SiNc shows essentially no selectivity, since the photosensitizer concentrations in the skin (peritumoral tissue) were very similar to those found in the tumor at all postinjection times examined by us. Irradiation of SiNc-loaded melanoma with 776 nm light from a diode laser at 24 h postinjection induces tumor necrosis and delay of tumor growth. The effect appears to be of purely photochemical nature at dose rates up to 260 mW cm-2; at higher dose rates, thermal effects are likely to become important.

A variety of 1,8(11),15(18),22(25)- and 2,9(10),16(17),23(24)-tetra-substituted and 2,3,9,10,16,17,23,24-octa-susbtituted Zn (II) phthalocyanines, bearing dialkylaminophenoxy and trialkylammoniumphenoxy groups, have been synthetized, characterized and tested as photosensitizers against S. aureus, E. coli and C. albicans. All phthalocyanines exhibited intense absorption bands in the phototherapeutically useful 670-700 nm region, with a molar extinction coefficient of the order of 105 M-1.cm-1. All derivatives bearing positive charges showed a good photosensitizing efficiency (causing a greater than 4 log reduction in cell survival), as well as a high quantum yield for generation of singlet oxygen. Variations of the chemical structure appeared to strongly affect both the physicochemical properties and the phototoxic activities against microorganisms.