Delivery of a hydrophobic phthalocyanine photosensitizer using PEGylated gold nanoparticle conjugates for the in vivo photodynamic therapy of amelanotic melanoma.

Photodynamic therapy (PDT) is a treatment of cancer whereby tumours are destroyed by reactive oxygen species generated upon photoactivation of a photosensitizer drug. Hydrophobic photosensitizers are known to be ideal for PDT; however, their hydrophobicity necessitates that they are typically administered using emulsions. Here, a delivery vehicle for photodynamic therapy based on the co-self-assembly of both a Zn(ii)-phthalocyanine derivative photosensitizer and a polyethylene glycol (PEG) derivative onto gold nanoparticles is reported. The PEG on the particle surface ensured that the conjugates were water soluble and enhanced their retention in the serum, improving the efficiency of PDT in vivo. The pharmacokinetic behaviour of the nanoparticle conjugates following intravenous injection into C57/BL6 mice bearing a subcutaneous transplanted B78H1 amelanotic melanoma showed a significant increase of retention of the nanoparticles in the tumour. PDT tumour destruction was achieved 3 h following injection of the nanoparticle conjugates leading to a remarkable 40% of the treated mice showing no tumour regrowth and complete survival. These results highlight that dual functionalised nanoparticles exhibit significant potential in PDT of cancer especially for difficult to treat cancers such as amelanotic melanoma.

Synthesis and photophysical properties of the photoactivatable cationic porphyrin 5-(4-N-dodecylpyridyl)-10,15,20-tri(4-N-methylpyridyl)-21H,23H-porphyrin tetraiodide for anti-malaria PDT.

This article describes a new synthetic method for obtaining three water soluble porphyrins. The more sophisticated porphyrin [5-(4-N-dodecylpyridyl)-10,15,20-tri(4-N-methylpyridyl)-21H,23H-porphyrin tetraiodide], also named C12 porphyrin, was obtained through a three step methodology. The improvements, compared to syntheses described in the literature, mostly concern the purification procedures. The photophysical properties of the three porphyrins are described and the C12 porphyrin presents a very good (1)O2 yield compared to its chemical intermediates. This porphyrin seems to be a very promising candidate for PDT applications.

Porphyrin-silica microparticle conjugates as an efficient tool for the photosensitised disinfection of water contaminated by bacterial pathogens.

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.

Effects of a new photoactivatable cationic porphyrin on ciliated protozoa and branchiopod crustaceans, potential components of freshwater ecosystems polluted by pathogenic agents and their vectors.

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.

Alterations of Escherichia coli envelope as a consequence of photosensitization with tetrakis(N-ethylpyridinium-4-yl)porphyrin tetratosylate.

Although several details of the photosensitization mechanisms involved in the photosensitized inactivation of bacteria have been elucidated, there are relatively few data on the morphological alterations induced on the bacterial cell structure during photosensitization. In this work we analysed the photodynamic action of the tetra-cationic photosensitizer tetrakis(N-ethylpyridinium-4-yl)porphyrin tetratosylate (TN-Et-PyP) on the integrity and selected functions of E. coli KMY1 cell membranes, in an effort to combine electron microscopy data with enzymatic assays and electrochemistry measurements. Using low concentrations of photosensitizer, damage is inflicted to the outer membrane and results in a higher permeability of the membrane to fairly small molecules such as deoxycholate; however, larger molecules such as periplasmic alkaline phosphatase are not released or are released after their extensive inactivation, as we could not register any enzyme activity outside the cells. Increasing the TN-Et-PyP concentration correlates with the inactivation of the respiratory chain, drop in plasma membrane voltage, the release of compounds with absorption band at 260 nm, and a decrease in intracellular enzyme ß-galactosidase activity, though this activity has not been noticed to increase outside the cells, suggesting that enzyme inactivation probably occurs in inner cell districts.

Switch from inhibition to activation of the mitochondrial permeability transition during hematoporphyrin-mediated photooxidative stress. Unmasking pore-regulating external thiols.

We have studied the mitochondrial permeability transition pore (PTP) under oxidizing conditions with mitochondria-bound hematoporphyrin, which generates reactive oxygen species (mainly singlet oxygen, (1)O(2)) upon UV/visible light-irradiation and promotes the photooxidative modification of vicinal targets. We have characterized the PTP-modulating properties of two major critical sites endowed with different degrees of photosensitivity: (i) the most photovulnerable site comprises critical histidines, whose photomodification by vicinal hematoporphyrin causes a drop in reactivity of matrix-exposed (internal), PTP-regulating cysteines thus stabilizing the pore in a closed conformation; (ii) the most photoresistant site coincides with the binding domains of (external) cysteines sensitive to membrane-impermeant reagents, which are easily unmasked when oxidation of internal cysteines is prevented. Photooxidation of external cysteines promoted by vicinal hematoporphyrin reactivates the PTP after the block caused by histidine photodegradation. Thus, hematoporphyrin-mediated photooxidative stress can either inhibit or activate the mitochondrial permeability transition depending on the site of hematoporphyrin localization and on the nature of the substrate; and selective photomodification of different hematoporphyrin-containing pore domains can be achieved by fine regulation of the sensitizer/light doses. These findings shed new light on PTP modulation by oxidative stress.

Inclusion of 5-[4-(1-dodecanoylpyridinium)]-10,15,20-triphenylporphine in supramolecular aggregates of cationic amphiphilic cyclodextrins: physicochemical characterization of the complexes and strengthening of the antimicrobial photosensitizing activity.

Recent findings suggest that visible light-promoted photooxidative processes mediated by sensitizers of appropriate chemical structure could represent a useful tool for properly addressing the problem of the increasing occurrence of infectious diseases caused by multiantibiotic-resistant microbial pathogens. The monocationic meso-substituted porphyrin 5-[4-(1-dodecanoylpyridinium)]-10,15,20-triphenyl-porphine (TDPyP) complexed into supramolecular aggregates of cationic amphiphilic beta-cyclodextrin (SC(6)NH(2)) (mean diameter = 20 nm) appeared to be endowed with favorable properties to act as a photosensitizing agent, including a very high quantum yield (Phi(Delta) = 0.90) for the generation of the highly reactive oxygen species, singlet oxygen ((1)O(2)). Although the yield of (1)O(2) generation was comparable to that obtained after TDPyP incorporation into cationic unilamellar liposomes of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) SC(6)NH(2)-bound TDPyP was more active than DOTAP-bound TDPyP in photosensitizing the inactivation of the Gram-positive methicillin-resistant bacterium Staphylococcus aureus (MRSA). At variance with DOTAP-bound TDPyP, photoactivated SC(6)NH(2)-bound TDPyP was efficient also in photokilling Gram-negative bacterial pathogens, such as Escherichia coli . These observations are in agreement with the well-known photobactericidal effect of positively charged porphyrin derivatives, which can be markedly enhanced after incorporation into carriers with multiple positive charges. In addition, transmission electron microscopy studies revealed that potentiation of the TDPyP-mediated photobactericidal effect by incorporation into SC(6)NH(2) is a consequence of the carrier's ability to promote an efficient crossing of the very tightly organized three-dimensional architecture of the bacterial outer wall by the embedded porphyrin so that a prompt interaction between the short-lived photogenerated (1)O(2) and the nearby targets, whose integrity is critical for cell survival, can take place.

Photothermal sensitisation and therapeutic properties of a novel far-red absorbing cyanine.

A water-soluble disulfonate cyanine was prepared by chemical synthesis and shown to possess photophysical properties which are particularly favourable for the promotion of photothermally sensitised processes, including a very low (<0.1) quantum yield of fluorescence emission and ultra-short (110 to 400 ps) excited state lifetimes, as well as the presence of intense absorption bands at wavelengths longer than 800 nm. This allows the possibility of high-energy irradiation by means of a Ti:sapphire laser operated in a pulse regime. The cyanine was accumulated in comparable amounts by B78H1 amelanotic melanoma cells and HT1080 transformed fibroblasts, however only the B78H1 cells could be extensively damaged by photothermal sensitisation with the cyanine, which was endocellularly distributed as suggested by observations at the optical microscope; the efficiency of the photoprocess could be enhanced by formation of aggregated intracellular cyanine clusters. On the other hand, only a modest photoinactivation of HT1080 cells was induced by photothermal sensitisation, possibly owing to the localization of the cyanine at the periphery of such cells. The cyanine also exhibited a good selectivity of amelanotic melanoma targeting in C57BL/6 mice, bearing the tumour subcutaneously transplanted in the dorsal area: the ratio of cyanine concentration in the melanoma and the surrounding cutaneous districts was as large as 3.8 at 1 h post-injection. The cyanine underwent a fast clearance from the organism, since only traces of the photosensitiser were observed in all the studied tissues at 3 h after i.v. administration. Thus, irradiations were performed at post-injection times shorter than 1 h. Maximum photothermal sensitisation efficiency was obtained at 10 min after injection with a 50% cure rate. Thus, photothermal therapy (PTT) appears to be a very promising and efficient modality of tumour treatment.

Metallo-naphthalocyanines as photothermal sensitisers for experimental tumours: in vitro and in vivo studies.

BACKGROUND AND OBJECTIVES: Photothermal sensitisation has been recently proposed as a novel approach for the treatment of solid tumours through the development of a therapeutic modality named photothermal therapy (PTT). The technique involves the use of high power pulsed laser irradiation and photosensitising agents with especially short lifetime (in the subnanosecond range) in the electronically excited states. This study aims to investigate the molecular features of the photosensitiser which optimise the photothermal activity. STUDY DESIGN/MATERIALS AND METHODS: Two octabutoxy-naphthalocyanines centrally coordinated with Pd(II) or Pt(II) ions were prepared by chemical synthesis and tested for their affinity and photothermal sensitisation activity toward a selected tumour cell line, namely B78H1 amelanotic melanoma. Irradiations were performed by using a Ti:sapphire laser operated in a pulsed regime (10 Hz, 30 nanosecond pulses, 120 mJ) at 809 nm (Pt) or 826 nm (Pd). The subcellular distribution pattern of the photosensitiser was also assessed by optical microscopy, while the nature of the photoinduced cell damage was determined by scanning electron microscopy. The results thus obtained provided a basis for subsequent in vivo studies, aimed at defining the phototherapeutic efficiency of the two metallo-naphthalocyanines: the photosensitisers were i.v. injected into C57BL/6 mice bearing a subcutaneously transplanted amelanotic melanoma and at 24 hours post-injection the tumour area was irradiated by the Ti:sapphire laser using the same protocol as above detailed. RESULTS: Both naphthalocyanines exhibited a high affinity for the amelanotic melanoma cells. The subcellular distribution pattern was modulated by the incubation time: after 48 hours incubation with 7.7 microM Pd- and Pt derivatives, the naphthalocyanine appeared to localise in specific sites with a gradual formation of aggregated clusters. Subsequent irradiation of the naphthalocyanine-loaded cells caused an extensive cell death; the photoinduced damage, as observed at the scanning electron microscope, mainly consisted in the formation of large endocellular holes consequent to the loss of cytoplasmic material. This scenario is typical of photothermal sensitisation processes. Lastly, both metallo-naphthalocyanines, and in particular the Pd(II) derivative, promoted an important response by the amelanotic melanoma, when the neoplastic tissue was irradiated by the pulsed Ti:sapphire laser. In certain cases, the photothermal treatment appeared to be curative. In all cases, the in vivo photodamage was confined within the tumour area with no detectable involvement of the perilesional tissues. CONCLUSION: PTT appears to act very efficiently at least on subcutaneous tumours. The technique can be used either in combination with photodynamic therapy (PDT) or as an alternative to PDT in those cases where the latter modality displays a limited efficacy, such as in the treatment of pigmented or poorly vascularised tumours.

Synthesis and biological investigations of tetrakis(p-carboranylthio-tetrafluorophenyl)chlorin (TPFC).

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.

Efficient photoinactivation of methicillin-resistant Staphylococcus aureus by a novel porphyrin incorporated into a poly-cationic liposome.

Antimicrobial photodynamic therapy is emerging as a promising therapeutic modality for bacterial infections. Our studies aim at identifying strategies for optimizing the antibacterial activity of porphyrin-type photosensitisers. The photoinactivation properties of a novel, positively charged meso-substituted porphyrin, namely 5-[4-(1-dodecanoylpyridinium)]-10,15,20-triphenyl-porphyrin were tested against a typically antibiotic-resistant pathogen, such as methicillin-resistant Staphylococcus aureus. This porphyrin is characterized by an unusually large quantum yield (0.95) for the generation of the hyper-reactive oxygen species, singlet oxygen. In spite of this, it exhibits a relatively low photosensitising activity against bacteria when dissolved in a homogeneous aqueous solution or incorporated into neutral lipid vesicles. On the contrary, a dramatic potentiation of the photocydal effect takes place when polycationic agents such as liposomes of N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride are used as carriers. The cationic carrier primarily acts as a disorganizing agent for the native three-dimensional architecture of the bacterial wall, thereby enhancing its permeability to the photosensitiser. Consequently, the drug can deeply penetrate into the plasma membrane, and rapidly impair selected enzymic activities leading to cell death. Thus, the combination of positively charged drugs and cationic delivery systems appears to represent an innovative modality for achieving an efficient antimicrobial activity and opens new avenues for the development of this phototherapeutic application.

Tumour-localizing and -photosensitising properties of meso-tetra(4-nido-carboranylphenyl)porphyrin (H2TCP).

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.

Photodynamic therapy of microbial infections: state of the art and perspectives.

Photodynamic therapy (PDT) is coming of age as an efficient alternative treatment for microbial infections, a problem which is presently aggravated by the increasingly widespread diffusion of antibiotic-resistant microbial strains. In particular, the use of red light-absorbing photosensitizers as photodynamic antimicrobial agents is characterized by various favorable features, including: (a) the broad spectrum of antimicrobial action of selected phenothiazines, porphyrins, and phthalocyanines, which promote the photosensitized inactivation of Gram(+) and Gram(-) bacteria, fungi, mycoplasma, and parasites by using one phototherapeutic protocol and mild irradiation conditions; (b) porphyrins/phthalocyanines display no appreciable toxicity in the dark at photochemically active doses; (c) microbial cell death is primarily a consequence of membrane photodamage through a typically multitarget process, which minimizes the risk of both the onset of mutagenic processes and the selection of photoresistant cells; (d) such photosensitizers act with essentially identical efficiency against both wild and antibiotic-resistant strains, whereas no selection of photoresistant microbial pathogens has been observed; (e) a combination between antibiotic-based and photodynamic therapy is possible. A typical example of phthalocyanine-sensitized photoinactivation of methicillin-resistant Staphylococcus aureus (MRSA) is provided. At present, antimicrobial PDT appears to be especially convenient for the treatment of localized infections, such as oral candidosis, periodontitis or chronic wounds.

Inactivation of methicillin-resistant Staphylococcus aureus (MRSA) by liposome-delivered photosensitising agents.

The uptake of two photosensitising agents (hematoporphyrin and chlorophyll a) by a highly pathogenic bacterium, namely methicillin-resistant Staphylococcus aureus (MRSA), has been studied by using unilamellar liposomes of different size, fluidity and electric charge as carriers. Optimal results are obtained by using hematoporphyrin embedded in fluid cationic vesicles composed by the monocationic lipid N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), which yield an endocellular concentration of photosensitiser much higher than that obtained by incubation of the cells with the free porphyrin, yet promote a tighter binding and a more efficient photoinactivation of MRSA. Apparently, the photosensitiser is successfully transferred from the liposome to the bacterial cells when the presence of the tetrapyrrolic derivative does not appreciably perturb the native three-dimensional organisation of the lipid vesicle, such as it occurs with hematoporphyrin. On the other hand, chlorophyll, which causes a marked structural alteration of the DOTAP vesicles as shown by electron microscopy and fluorescence anisotropy measurements, does not show any detectable photocytotoxicity toward MRSA, contrary to what observed for the free dye.

Inactivation of vascular smooth muscle cells photosensitised by liposome-delivered Zn(II)-phthalocyanine.

Photodynamic therapy is a promising approach for the prevention of arterial restenosis, which frequently occurs after balloon angioplasty, largely owing to abnormal proliferation of vascular smooth muscle cells (VSMC) and their migration from the media to the intima, where they originate intimal hyperplasia (IH). We investigated the efficacy of Zn(II)-phthalocyanine-photosensitised processes in promoting the inactivation of VSMC. This liposome delivered phthalocyanine is readily taken up by VSMC, largely partitions in the Golgi apparatus, and upon photoactivation causes >95% cell mortality using mild irradiation conditions (e.g. 5 min irradiation at 1 microM ZnPc). Cell death occurs through the parallel development of random necrotic and apoptotic processes.

Zn(II)-phthalocyanines as phototherapeutic agents for cutaneous diseases. Photosensitization of fibroblasts and keratinocytes.

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 microM 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/cm2 in the presence of 5 microM 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.

Discrimination between two steps in the mitochondrial permeability transition process.

It is well known that a lag phase generally elapses between the addition of inducers of the mitochondrial permeability transition and the opening of the pore. To advance our present understanding as regards the significance of this phenomenon, we used experimental approaches which are sensitive to different aspects of the permeability transition process. The pore conformation was sensed by the fluorescence anisotropy changes of hematoporphyrin-labelled mitochondria. Membrane permeabilization was ascertained by following the matrix swelling consequent to external solute equilibration. We show that the anisotropy changes of mitochondria-bound hematoporphyrin precede both membrane depolarization (proton permeation) and matrix swelling (solute permeation), thus sensing a step of the permeability transition process that involves the pore in its closed state. We suggest that the opening of the pore is preceded by a structural remodelling of mitochondrial domains containing hematoporphyrin-near, pore-regulating histidines. Such a perturbation is strongly inhibited at acidic matrix pH and completely blocked by cyclosporin A. In sucrose-based media the opening of the pore can be strongly delayed, as compared to salt-based media, a fact which probably reflects perturbation of mitochondrial membranes by sugar. We conclude that the mitochondrial permeability transition could be described as an at least two-step process which is mainly regulated by conformational changes of the pore components.

Photothermal sensitisation as a novel therapeutic approach for tumours: studies at the cellular and animal level.

Irradiation of B78H1 murine amelanotic melanoma cells with 850 nm light emitted from a Ti:sapphire laser, operated in a pulsed mode at high fluence rates and in the presence of Ni(II)-octabutoxy-naphthalocyanine (NiNc), promoted a photothermally sensitised process leading to fast and irreversible cell death. This resulted in the ejection of a consistent mass of cytoplasmic material from the irradiated cells that was detected by scanning electron microscopy. The extensive chemical and mechanical damage was probably caused by the photoinduced generation of an acoustic shock wave. The efficiency of the photoprocess was modulated by intracellular concentration of NiNc and maximally by the formation of aggregated naphthalocyanine clusters in specific subcellular areas. Very similar results were obtained upon irradiation of NiNc-loaded C32 human amelanotic melanoma cells and transformed murine HT-1080 and HaCaT fibroblasts. From these results, photothermal sensitisation appears to be a general phenomenon and preliminary studies with mice bearing subcutaneously transplanted amelanotic melanomas, irradiated with 850 nm light 24 h after intravenous injection of NiNc, suggest that this approach has potential for the therapy of some types of skin tumours.

Low-density lipoprotein receptors in the uptake of tumour photosensitizers by human and rat transformed fibroblasts.

Low-density lipoproteins (LDL) increase the selectivity of tumour targeting by drugs, including sensitisers for photodynamic therapy, because of the enhanced expression of specific LDL receptors in many types of transformed as compared with non-transformed cells. This investigation aims at gaining more information on the role of LDL receptors in the accumulation of photosensitizer-LDL complexes by human and rat transformed fibroblasts, and the interference of the photosensitizer with LDL recognition by the specific receptors. Both an amphiphilic hematoporphyrin IX (Hp) and a hydrophobic Zn(II)-phthalocyanine (ZnPc) photosensitizers bind to human LDL with molar ratios of 5-6:1 and 10-12:1, respectively. The hematoporphyrin-LDL complex is accumulated by human HT1080 fibroblasts mainly through the high affinity LDL receptors, while the Zn-phthalocyanine-LDL complex is internalised through non specific endocytosis because of changes in the apoB LDL structure induced by phthalocyanine association, as suggested by spectroscopic studies. The uptake of LDL-delivered hematoporphyrin, but not Zn-phthalocyanine, is about 4-fold higher in HT1080 cells stimulated for maximal expression of LDL receptors as compared with non-stimulated cells. This difference is abolished by LDL acetylation. Human LDL-bound hematoporphyrin and Zn-phthalocyanine are up taken by stimulated and non-stimulated 4R rat fibroblasts with similar efficiency. Scatchard plot analysis of human (125)I-LDL binding to 4R cells shows the presence of only low affinity receptors while 350,000 high affinity receptors are expressed per HT1080 cell. It is concluded that a careful evaluation of the lack of conformational changes of LDL is critical for guaranteeing the selectivity and efficiency of photosensitizer delivery to tumour cells.

Synthesis and antibacterial activity of new poly-S-lysine-porphyrin conjugates.

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.