Folate-targeted PEGylated liposomes improve the selectivity of PDT with meta-tetra(hydroxyphenyl)chlorin (m-THPC).

The folate receptor (FR) is over-expressed in many human tumours and is being intensively studied also in the field of nanomedicine as a target to enhance the selectivity of drug delivery to cancer cells by using nanocarriers bearing folic acid (FA) on their surface. In this study we report the encapsulation of the photosensitizer (PS) meta-tetra(hydroxyphenyl)chlorin (m-THPC) in FA-targeted PEGylated liposomes used as a novel drug delivery system for photodynamic therapy (PDT) of cancer. Our in vitro investigations revealed that only a modest fraction of targeted liposomes were internalized by specific endocytosis in FR-positive KB cells. However, FA-liposomes doubled the uptake of the entrapped m-THPC with respect to un-targeted liposomes and enhanced the photo-induced cytotoxicity in KB cells. In contrast, in FR-negative A549 cells FA-targeted or un-targeted liposomes exhibited a very similar extent of internalization and as a consequence the same photo-killing efficiency.

Effect of light exposure on metalloporphyrin-treated newborn mice.

BACKGROUND: Neonatal hyperbilirubinemia arises from increased bilirubin production and decreased bilirubin elimination. Although phototherapy safely and effectively reduces bilirubin levels, recent evidence shows that it has adverse effects. Therefore, alternative treatments are warranted. Metalloporphyrins, competitive inhibitors of heme oxygenase (HO), the rate-limiting enzyme in bilirubin production, effectively reduce bilirubin formation; however, many are photoreactive. Here, we investigated possible photosensitizing effects of chromium mesoporphyrin (CrMP) and zinc deuteroporphyrin bis-glycol (ZnBG). METHODS AND RESULTS: Administration of CrMP or ZnBG to 3-d-old mouse pups (3.75-30.0 µmol/kg intraperitoneally) and exposure to cool white (F20T12CW) and blue (TL20W/52) fluorescent lights (+L) for 3 h, resulted in a dose-dependent mortality (50% lethal dose (LD50) = 21.5 and 19.5 µmol/kg, respectively). In contrast to ZnBG, there was no significant difference in survival between the CrMP+L and CrMP groups. Following 30 µmol/kg ZnBG+L, we found significant weight loss, decreased liver antioxidant capacities, and increased aspartate aminotransaminase levels. At 6-d post-light exposure, ZnBG+L-treated pups showed gross and histologic skin changes at doses >7.5 µmol/kg. No lethality was observed following treatment with 30 µmol ZnBG/kg plus exposure to blue light-emitting diodes. Phototoxicity of ZnBG was dependent on light source, emission spectrum, and irradiance. CONCLUSION: Low doses of ZnBG (<3.75 µmol/kg) retained maximal HO inhibitory potency without photosensitizing effects, and therefore are potentially useful in treating neonatal hyperbilirubinemia.

Meta-tetra(hydroxyphenyl)chlorin-loaded liposomes sterically stabilised with poly(ethylene glycol) of different length and density: characterisation, in vitro cellular uptake and phototoxicity.

We studied the effects of density and thickness of PEG coating on in vitro cellular uptake, and dark- and photo-toxicity of liposomal formulations (Fospeg) of the photodynamic agent meta-tetrahydroxyphenyl chlorin (m-THPC). The cellular uptake of various Fospeg formulations was determined by flow cytometry in CCD-34Lu human normal fibroblasts and A549 lung cancer cells. Dark and light-induced cytotoxicity was measured by MTS assay after exposure to increasing concentrations of Fospeg only and followed by irradiation with red light. Intracellular localization of m-THPC delivered by Fospeg was determined by fluorescence microscopy. The studies were carried out in comparison with m-THPC delivered by the standard solvent. In the dark all Fospeg formulations were less cytotoxic than m-THPC in standard solvent (ethanol/poly(ethylene glycol 400/water; 20 : 30 : 50 by vol.) and cytotoxicity decreased by increasing PEGylation. m-THPC delivered as Fospeg was internalised by endocytosis and localised mainly in the Golgi apparatus and endoplasmic reticulum. The efficiency of cellular uptake of Fospeg was reduced by 30-40% with respect to m-THPC in standard solution causing a slight reduction of the phototoxicity but without serious impairment of the efficacy of the treatment. Our study suggests that PEGylated liposomes are promising nanocarriers for the delivery of photosensitisers for photodynamic therapy because they reduce dark cytotoxicity while preserving therapeutic efficacy.

Comparison of intracellular accumulation and cytotoxicity of free mTHPC and mTHPC-loaded PLGA nanoparticles in human colon carcinoma cells.

The second generation photosensitizer mTHPC was approved by the European Medicines Agency (EMA) for the palliative treatment of advanced head and neck cancer in October 2001. It is known that mTHPC possesses a significant phototoxicity against a variety of human cancer cells in vitro but also exhibits dark toxicity and can cause adverse effects (especially skin photosensitization). Due to its poor water solubility, the administration of hydrophobic photosensitizer still presents several difficulties. To overcome the administration problems, the use of nanoparticles as drug carrier systems is much investigated. Nanoparticles based on poly(lactic-co-glycolic acid) (PLGA) have been extensively studied as delivery systems into tumours due to their biocompatibility and biodegradability. The goal of this study was the comparison of free mTHPC and mTHPC-loaded PLGA nanoparticles concerning cytotoxicity and intracellular accumulation in human colon carcinoma cells (HT29). The nanoparticles delivered the photosensitizer to the colon carcinoma cells and enabled drug release without losing its activity. The cytotoxicity assays showed a time- and concentration-dependent decrease in cell proliferation and viability after illumination. However, first and foremost mTHPC lost its dark toxic effects using the PLGA nanoparticles as a drug carrier system. Therefore, PLGA nanoparticles are a promising drug carrier system for the hydrophobic photosensitizer mTHPC.

Photodynamic therapy of HeLa cell cultures by using LED or laser sources.

The photodynamic therapy (PDT) on HeLa cell cultures was performed utilizing a 637nm LED lamp with 1.06W power and m-tetrahydroxyphenyl chlorin (m-THPC) as photosensitizer and compared to a laser source emitting at 654nm with the same power. Intracellular placement of the photosensitizer and the effect of its concentration (CP), its absorption time (TA) and the illumination time (TI) were evaluated. It was observed that for CP>40µg/ml and TA>24h, m-THPC had toxicity on cells in culture, even in the absence of illumination. For the other tested concentrations, the cells remained viable if not subjected to illumination doses. No effect on cells was observed for CP<0.05µg/ml, TA=48h and TI=10min and they continued proliferating. For drug concentrations higher than 0.05µgml(-1), further deterioration is observed with increasing TA and TI. We evaluated the viability of the cells, before and after the treatment, and by supravital dyes, and phase contrast and fluorescence microscopies, evidence of different types of cell death was obtained. Tetrazolium dye assays after PDT during different times yielded similar results for the 637nm LED lamp with an illuminance three times greater than that of the 654nm laser source. Results demonstrate the feasibility of using a LED lamp as alternative to laser source. Here the main characteristic is not the light coherence but achieving a certain light fluence of the appropriate wavelength on cell cultures. We conclude that the efficacy was achieved satisfactorily and is essential for convenience, accessibility and safety.

Uptake and photo-toxicity of Foscan®, Foslip® and Fospeg® in multicellular tumor spheroids.

In cancer photodynamic therapy (PDT), an efficient and homogeneous intratumoral accumulation of the photosensitizer (PS) is required to induce cell damages in the entire tumor mass after light activation. Thus, in this study we investigated penetration ability and photodynamic efficiency of meta-tetra(hydroxyphenyl)chlorin (m-THPC) in standard formulation (Foscan®) and in its non PEGylated and PEGylated liposomal formulations, Foslip® and Fospeg®, in HeLa multicellular spheroids, as in vitro avascular models of solid tumors. Confocal microscopy studies demonstrated that m-THPC fluorescence was confined in the external cell layers of spheroids with a slightly higher accumulation of Foslip® and Fospeg® with respect to Foscan®. Irradiation with red light, following 24h incubation of spheroids with the m-THPC formulations, caused however photodamages also in cells located in the central part of spheroids, as documented by transmission electron microscopy analyses. Overall, the photodynamic effects of the three m-THPC formulations on HeLa cell spheroids were comparable in terms of cell viability measured with the MTS assay. It is however worth noting that the delivery of m-THPC by liposomes significantly abolished its cytotoxicity in the dark, slightly improved the cellular uptake and, following PDT, promoted cell loss and spheroid disassembling to a higher extent when compared to Foscan®.

Light-induced retinal vascular damage by Pd-porphyrin luminescent oxygen probes.

PURPOSE: The phosphorescence lifetime of certain metalloporphyrins dissolved in a physiological medium provides an optical signature for local oxygen concentration (pO(2)). This effect is used for measuring physiological pO(2) levels in various tissues. However, the phosphorescence quenching of certain metalloporphyrin triplet states by oxygen also creates singlet oxygen, which is highly reactive and capable of inducing tissue damage. In the current study, the Pd-meso-tetra(4-carboxyphenyl) porphyrin dye (PdTCPP) was simultaneously used as an oxygen sensor and a photosensitizer. Phototoxicity was assessed in the eye fundus and correlated with tissue oxygenation, drug-light dose, and severity of tissue damage. METHODS: The kinetics of photochemical oxygen depletion during PdTCPP excitation was measured in vivo on the optic disc of piglets by phosphorescence lifetime imaging. Blood-retinal barrier breakdown and tissue damage were assessed by confocal and electron microscopy. RESULTS: For a retinal irradiance of 5 mW/cm(2) at 532 nm and an injected PdTCPP dose of 20 mg/kg, the mean phosphorescence lifetime measured at the optic disc increased from 100 to 600 micros within 8 minutes of continuous illumination. This corresponds to a decrease of pO(2) from 25 to 0 mm Hg, induced by a light dose of only 2.4 J/cm(2). An exposure time of 6 minutes (1.8 J/cm(2)) generated an increase in phosphorescence lifetime from 100 to 400 micros, corresponding to a decrease in pO(2) from 25 to 4 mm Hg. This caused edema in all retinal layers, whereas irradiation of 2 minutes (0.6 J/cm(2)) damaged blood vessels and induced edema in the inner nuclear layer only. Heavy redistribution of occludin occurred after a 30-minute exposure time (9 J/cm(2)). CONCLUSIONS: PdTCPP is potentially phototoxic under certain experimental conditions and can induce damage in peripapillary retina and optic nerve head after light exposure. The severity of tissue damage correlates with the phosphorescence measurements.

Specific fluorescent tracers. Imaging and applications for photodynamic therapy.

Our main objective is to enlarge the fluorescence use in biosciences, with especially the photodynamic therapy (PDT) used for cancer treatment as one of the target applications. Meta-tetra(hydroxyphenyl)chlorin (m-THPC) is a second-generation photosensitiser, applied in photodynamic therapy. The localisation of this sensitiser as well as its induced cell death mechanisms in human breast cancer cells (MCF-7 and its resistant subline MCF-7DXR, DXR: doxorubicin) were evaluated using fluorescence microscopy. In addition, we will present two additional routes, whose aims are to create new features to respond to the PDT questioning: firstly, the synthesis of fluorescent tracers, with a particular attention to the presence of hydrophilic groups (glucosamine ring) on the basic fluorophore structure to orientate the localisation of the probe and, secondly, the use of scanning near-field optical microscopy to reach a better resolution for the fluorescence microscopy analysis.

Isobolographic assessment of the interaction between adriamycin and photodynamic therapy with meso-chlorin e6 monoethylene diamine in human epithelial ovarian carcinoma (OVCAR-3) in vitro.

OBJECTIVE: Considering the differing mechanisms of cytotoxicity produced by adriamycin and the photosensitizer meso-chlorin e6 monoethylene diamine (Mce6) with light, the interaction of these agents in combination on human ovarian epithelial carcinoma (OVCAR-3 in vitro) was evaluated by dose and effect addition isobole analysis. METHODS: Mitochondrial respiration via the 3-(4,5-dimethyl thiazol-2 yl)-2,5-diphenyl tetrazolium bromide cleavage assay (MTT) and reproductive capacity via the tritiated thymidine incorporation assay (TI) were assessed 72 and 144 hours after exposure to adriamycin, Mce6, and light (650 nm), and to their combinations, in OVCAR-3 cells grown in vitro (20,000 cells per well). RESULTS: In the majority of assays, reproductive capacity was more sensitive to the drug(s) than was mitochondrial respiration (2-10x). Dose-addition isobole analysis showed synergy for the combination of 50% median effective dose (ED50) adriamycin with 50% ED50 Mce6/light in all assays (all P < or = .027). Antagonism was noted with the combination 25% ED50 adriamycin with 75% ED50 Mce6/light. Additivity and synergy were the predominant interactions for 75% ED50 adriamycin with 25% ED50 Mce6/light by dose-addition isobole analyses. Effect-addition isoboles showed a predominance of synergy, particularly for the combination 50% ED50 adriamycin with 50% ED50 Mce6/light. CONCLUSION: Synergy and additivity are the primary in vitro interactions for the combination of adriamycin and Mce6/light in the dosage range tested. Reproductive capacity is more sensitive to these agents than is mitochondrial respiration.

Clinical evaluation of the cutaneous phototoxicity of 5,10,15,20-tetra(m-hydroxyphenyl)chlorin.

The cutaneous phototoxic reaction induced by intravenous injection of 5,-10,-15,-20-tetra(m-hydroxyphenyl)chlorin (mTHPC) has been clinically evaluated in patients undergoing photodynamic therapy. These tests were performed on the backs of 23 patients with a solar simulator at various times after drug administration ranging from 5 h to 57 days. The mTHPC doses ranged from 0.1 to 0.3 mg/kg, and the illuminations lasted from 30 s up to 8 min. These tests have shown that the duration of the skin photosensitization induced after a typical therapeutic dose of mTHPC (0.15 mg/kg) is less important than with Photofrin (2 mg/kg). The level of mTHPC in the skin was also assessed in vivo and at times corresponding to the irradiations using an optical fiber-based spectrofluorometer. This study indicates that the light-induced fluorescence spectroscopy of mTHPC enables prediction of the degree of photosensitivity of the skin.

PEG-m-THPC-mediated photodynamic effects on normal rat tissues.

Photodynamic therapy (PDT) of malignancies uses light to activate a photosensitizer preferentially accumulated in cancer cells. The first pegylated photosensitizer, tetrakis-(m-methoxypolyethylene glycol) derivative of 7,8-dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)-21-23-[H]-porphyrin (PEG-m-THPC), was evaluated in non-tumor-bearing rats. The aim of this study was to assess the photodynamic threshold for damage and its sequelae in normal rat tissue. Thirty-five Fischer rats were sensitized with 3, 9 or 30 mg/kg body weight PEG-m-THPC. Colon, vagina and perineum were irradiated with laser light of 652 nm wavelength and an optical dose of 50, 150 or 450 J/cm fiber length. Temperature in the pelvis was measured during PDT. Three days following PDT the effect on skin, vagina, colon, striated muscle, connective tissue, nerves and blood vessels was assessed by histology. The healing of the above-mentioned tissues was assessed on two rats 3 and 8 weeks after PDT using 9 mg/kg PEG-m-THPC activated with 450 J/cm laser light. No dark toxicity was observed. PDT using 30 mg/kg PEG-m-THPC induced severe necrosis irrespective of the optical dose. Body weight of 9 or 3 mg/kg activated with less than 450 J/cm induced moderate or no damage. No substantial increase in body temperature was seen during PDT. Tissues with severe PDT-induced damage seem to have a good tendency to regenerate. We conclude that within the dose required for tumor treatment PEG-m-THPC is a safe photosensitizer with promising properties. PDT of the colon mucosa below 9 mg/kg PEG-m-THPC and 150 J/cm seems to be safe. All other tissues can be exposed to 9 mg/kg PEG-m-THPC activated with less than 450 J/cm laser light with little side effects.

m-THPC-mediated photodynamic therapy (PDT) does not induce resistance to chemotherapy, radiotherapy or PDT on human breast cancer cells in vitro.

Photodynamic therapy (PDT) uses laser light to activate a photosensitizer that has been absorbed preferentially by cancer cells after systemic administration. A phototoxic reaction ensues resulting in cell death and tissue necrosis. Some cells, however, may survive PDT. This study was performed to determine if surviving human breast cancer cells (MCF-7) can become resistant to PDT, chemotherapy or radiotherapy. The MCF-7 cells were cultured under standard conditions prior to being exposed to the photosensitizer, 5,10,15,20-meta-tetra(hydroxyphenyl)chlorin (m-THPC), for 24 h and then irradiated with laser light (652 nm). Surviving cells were allowed to regrow by allowing a 2 week interval between each additional PDT. After the third and final treatment, colony formation assays were used to evaluate the sensitivity of cultured cells to ionizing radiation and PDT and the ATP cell viability assay tested in vitro chemosensitivity. Flow cytometry was used to analyze the cell cycle. No alterations in the cell cycle were observed after three cycles of PDT with m-THPC. Similar responses to chemotherapy and ionizing radiation were seen in control and treatment groups. The m-THPC-sensitized PDT did not induce resistance to subsequent cycles of PDT, chemo- or radiotherapy. Photodynamic therapy with m-THPC may represent a novel adjunctive treatment of breast cancer that may be combined with surgery, chemotherapy or ionizing radiation.

The binding characteristics and intracellular localization of temoporfin (mTHPC) in myeloid leukemia cells: phototoxicity and mitochondrial damage .

The state of aggregation of the photosensitizer meso-tetrahydroxyphenylchlorin (mTHPC) in both cell free and intracellular environment was elucidated by comparing its absorption and excitation spectra. In methanol, mTHPC existed as monomers and strongly fluoresced. In aqueous solutions such as phosphate-buffered saline (PBS), mTHPC formed nonfluorescent aggregates. Some portion of mTHPC monomerized in the presence of 10% fetal calf serum PBS. In murine myeloid leukemia M1 and WEHI-3B (JCS) cells, cytoplasmic mTHPC were monomeric. By using organelle-specific fluorescent probes, it was found that mTHPC localized preferentially at the mitochondria and the perinuclear region. Photodynamic treatment of mTHPC-sensitized leukemia cells caused rapid appearance of the apoptogenic protein cytochrome c in the cytosol. Results from flow cytometric analysis showed that the release of cytochrome c was especially pronounced in JCS cells, and well correlated with the extent of apoptotic cell death as reported earlier. Electron microscopy revealed the loss of integrity of the mitochondrial membrane and the appearance of chromatin condensation as early as 1 h after light irradiation. We conclude that rapid release of cytochrome c from photodamaged mitochondria is responsible for the mTHPC-induced apoptosis in the myeloid leukemia JCS and M1 cells.

A comparison of the photodynamic effects of temoporfin (mTHPC) and MC540 on leukemia cells: efficacy and apoptosis.

The photodynamic effects of temoporfin (meso-tetrahydroxyphenylchlorin, mTHPC) and merocyanine 540 (MC540) in murine myeloid leukemia M1 and WEHI 3B (JCS) cells were compared. The mTHPC was found to be more potent and selective. At a lethal dosage of 90% killing (LD90), only 1.3 microM of mTHPC and 4.2 kJ/m2 of light irradiation was required, which was a 20-fold lower drug concentration and 11-fold smaller light dose than that required when using MC540. Meanwhile, three times less, or 15%, of the coincubated erythrocytes were destroyed by mTHPC than by MC540. Confocal micrographs showed that both drugs accumulated diffusely inside the cytoplasm in a very similar fashion, but mTHPC induced a more extensive apoptosis in photosensitized JCS cells. For example, at LD90, mTHPC practically killed all JCS cells via apoptosis and cleaved the DNA to extremely small 150 base-pair fragments. In contrast, among the JCS cells killed by MC540, about 88% died via apoptosis and large DNA fragments were abundant. Relative to MC540, the ability of mTHPC to trigger large-scale and thorough apoptosis in leukemia cells may help explain its potency and selectivity.

Comparative sensitivity of microvascular endothelial cells, fibroblasts and tumor cells after in vitro photodynamic therapy with meso-tetra-hydroxyphenyl-chlorin.

The phototoxic effect of meso-tetra-hydroxyphenyl-chlorin (mTHPC)-mediated photodynamic therapy (PDT) on human microvascular endothelial cells (hMVEC) was compared with that on human fibroblasts (BCT-27) and two human tumor cell lines (HMESO-1 and HNXOE). To examine the relationship between intrinsic phototoxicity and intracellular mTHPC content, we expressed cell survival as a function of cellular fluorescence. On the basis of total cell fluorescence, HNXOE tumor cells were the most sensitive and BCT-27 fibroblasts the most resistant, but these differences disappeared after correcting for cell volume. Endothelial cells were not intrinsically more sensitive to mTHPC-PDT than tumor cells or fibroblasts. Uptake of mTHPC in hMVEC increased linearly to at least 48 h, whereas drug uptake in the other cell lines reached a maximum by 24 h. No difference in drug uptake was seen between the cell lines during the first 24 h, but by 48 h hMVEC had a 1.8- to 2.8-fold higher uptake than other cell lines. Endothelial cells showed a rapid apoptotic response after mTHPC-mediated PDT, whereas similar protocols gave a delayed apoptotic or necrotic like response in HNXOE. We conclude that endothelial cells are not intrinsically more sensitive than other cell types to mTHPC-mediated PDT but that continued drug uptake beyond 24 h may lead to higher intracellular drug levels and increased photosensitivity under certain conditions.

Use of alkaline Comet assay to assess DNA repair after m-THPC-PDT.

Photodynamic therapy (PDT) with Photofrin has already been authorized for certain applications in Japan, the USA and France, and powerful second-generation sensitizers such as meta-(tetrahydroxyphenyl) chlorin (m-THPC) are now being considered for approval. Although sensitizers are likely to localize within the cytoplasm or the plasma membrane, nuclear membrane can be damaged at an early stage of photodynamic reaction, resulting in DNA lesions. Thus, it is of critical importance to assess the safety of m-THPC-PDT, which would be used mainly against early well-differentiated cancers. In this context, m-THPC toxicity and phototoxicity were studied by a colorimetric MTT assay on C6 cells to determine the LD50 (2.5 microg/ml m-THPC for 10 J/cm2 irradiation and 1 microg/ml for 25 J/cm2 irradiation) and PDT doses inducing around 25% cell death. Single-cell electrophoresis (a Comet assay with Tail Moment calculation) was used to evaluate DNA damage and repair in murine glioblastoma C6 cells after LD25 or higher doses for assays of PDT. These results were correlated with m-THPC nuclear distribution by confocal microspectrofluorimetry. m-THPC failed to induce significant changes in the Tail Moment of C6 cells in the absence of light, whereas m-THPC-PDT induced DNA damage immediately after irradiation. The Tail Moment increase was not linear (curve slope being 43 for 0-1 microg/ml m-THPC and 117 for 1-3 microg/ml), but the mean value increased with the light dose (0, 10 or 25 J/cm2) and incubation time (every hour from 1 to 4 h) for an incubation with m-THPC 1 microg/ml. However, cultured murine glioblastoma cells were capable of significant DNA repair after 4 h, and no residual DNA damage was evident after 24-h post-treatment incubation at 37 degrees C. An increase in the light dose appeared to be less genotoxic than an increase in the m-THPC dose for similar toxicities. Our results indicate that m-THPC PDT appears to be a safe treatment since DNA repair seemed to not be impaired and DNA damage occurred only with lethal PDT doses. However, the Comet assay cannot give us the certainty that no mutation, photoadducts or oxidative damage have been developed so this point would be verified with another mutagenicity assay.

Modes of photodynamic vs. sonodynamic cytotoxicity.

We compared effects of ultrasound-induced vs. photodynamic cytotoxicity in cell culture. The photodynamic effects mediated by mesoporphyrin caused a delayed toxic reaction, the presence of a "shoulder" on the dose-response curve, indicating the capacity for limited repair of photodamage. In contrast, ultrasound-induced loss of viability resulted from rapid cell destruction and was proportional to the time of sonication. Photodynamic damage to cells before exposure to ultrasound potentiated cell breakage but did not affect the clonogenicity of the surviving cell population. Photodamage after exposure to ultrasound decreased the viability of cells which had survived ultrasonic treatment.

A comparative study of the cellular uptake, localization and phototoxicity of meta-tetra(hydroxyphenyl) chlorin encapsulated in surface-modified submicronic oil/water carriers in HT29 tumor cells.

The poor selectivity of photosensitizers for tumor tissue remains a drawback in photodynamic therapy (PDT) and could be improved by adapted formulations. The cellular uptake, localization and phototoxicity of meta-tetra(hydroxyphenyl)chlorin (mTHPC) encapsulated in submicronic colloidal carriers have been studied in macrophage-like J774 cells and HT 29 human adenocarcinoma cells. Nanocapsules with an external layer made of poly(D,L lactic acid) (PLA NCs), PLA grafted with polyethylene glycol (PLA-PEG NCs), PLA coated with poloxamer 188 (polox PLA NCs) and oil/water nanoemulsion (NE) have been examined. The cellular uptake by J774, as determined by microspectroflorimetry, is reduced with mTHPC encapsulated into surface-modified NCs--PLA-PEG and polox PLA--compared with naked PLA, indicating a possible limitation of the clearance of such carriers by the reticuloendothelial system. Encapsulation also modifies the interaction between mTHPC and HT29 cells. Compared with the manufacturer's solution (PEG, ethanol, water), the cellular uptake is strongly reduced. However, the HT29 phototoxicity is much less affected and a protecting effect against plasma proteins is observed. Fluorescence microscopy reveals a specific punctate fluorescence pattern with PLA-PEG and polox PLA NCs in contrast to a more diffuse distribution with NE and solution, indicating that photodamage targeting could be different. These findings suggest that photosensitizers encapsulated into surface-modified nanocapsules could be a promising approach for improving PDT efficacy and this has to be confirmed in vivo.

Correlation between meta(tetrahydroxyphenyl)chlorin (m-THPC) biodistribution and photodynamic effects in mice.

Analysis of sensitizer kinetics is essential for the performance of light irradiation when tumour concentration and tumour-to-normal tissue ratios are optimal. In this study nude mice were grafted with human adenocarcinoma 15 days before meta(tetrahydroxyphenyl)chlorin (m-THPC) intra-peritoneal (IP) injection. Fluorescence was recorded through an optic fibre spectrofluorometer at 650 (the most intense) and 714 nm, with intensity being proportional to injected dose. In tumour, skin and muscle the maximum fluorescence was obtained with 1.6 mg kg-1 72 h after injection (44 counts per second). Tumour-to-skin and tumour-to-muscle ratios obtained by high performance liquid chromatography (HPLC) analysis and spectrofluorometric measurements decreased between 12 and 72 h (from 15 to 1.5), indicating that tumour selectivity decreases with time. This contrast between selectivity and fluorescence levels was also observed with photodynamic therapy (PDT) results, for which no differences were observed when 10 J cm-2 were delivered at 100 or 200 mW. PDT results were better 24 h after drug administration than at 72 h. Tumour growth decrease (-40%) was found when 1.6 mg kg-1 were injected 24 h before irradiation. For other groups a slower increase (12% vs. 23%) was noted in the first few days after PDT. The paradoxal correlation between fluorometric or HPLC measurements and PDT effects suggests that m-THPC localizes differently with time in tumour components.

Comparative in vitro study on the characteristics of different photosensitizers employed in PDT.

At present a wide range of photosensitizers are employed in photodynamic therapy (PDT) that have very different characteristics. Although, countless in vitro studies on the attributes of photosensitizers do exist, a direct comparison of these substances on one cell line are rare and may contribute to the choice of the optimal photoactive substance for a specific application. We therefore evaluated the properties of six widespread photosensitizers, namely Foscan, Fospeg, hypericin, aluminum (III) phthalocyanine tetrasulfonate chloride (AlPcS(4)), 5-aminolevulinic acid (ALA), and Photofrin in terms of: (i) cytotoxicity without illumination, (ii) phototoxicity, (iii) cellular uptake and release, and (iv) apoptosis induction in A431 human epidermoid carcinoma cells using comparable illumination regimens. We clearly show that meso-tetrahydroxyphenylchlorin (mTHPC, Foscan) is a very effective photosensitizer inducing high phototoxicity at very low concentrations. Similar in vitro characteristics and phototoxicity were observed for Fospeg, the water-soluble formulation of mTHPC. Hypericin, a photosensitizer extracted from plants of the Hypericum genus, is very effective in inducing apoptosis over a wide range of light fluences. AlPcS(4) absorbs light of 674 nm wavelength providing a higher penetration depth in tissue. Its hydrophilic character allows for application as aqueous solution. ALA can be administered at very high concentrations without producing cytotoxic effects in the dark. The intracellular concentration of protoporphyrin IX rapidly decreases after withdrawal of ALA, thus minimizing the period of light sensitivity post PDT. Among all photosensitizers Photofrin has most clinical approvals and serves as standard.