A nanochitosan-D-galactose formulation increases the accumulation of primaquine in the liver.

Primaquine is the mainstream antimalarial drug to prevent Plasmodium vivax relapses. However, this drug can induce hemolysis in patients with glucose-6-phosphate dehydrogenase deficiency. Nanostructure formulations of primaquine loaded with D-galactose were used as a strategy to target the drug to the liver and decrease the hemolytic risks. Nanoemulsion (NE-Pq) and nanochitosan (NQ-Pq) formulations of primaquine diphosphate containing D-galactose were prepared and characterized by their physicochemistry properties. Pharmacokinetic and biodistribution studies were conducted using Swiss Webster mice. A single dose of 10 mg/kg of each nanoformulation or free primaquine solution was administered by gavage to the animals, which were killed at 0.5, 1, 2, 4, 8, and 24 hours. Blood samples and tissues were collected, processed, and analyzed by high-performance liquid chromatography. The nanoformulation showed sizes around 200 nm (NE-Pq) and 400 nm (NQ-Pq) and physicochemical stability for over 30 days. Free primaquine solution achieved higher primaquine Cmax in the liver than NE-Pq or NQ-Pq at 0.5 hours. However, the half-life and mean residence time (MRT) of primaquine in the liver were three times higher with the NQ-Pq formulation than with free primaquine, and the volume distribution was four times higher. Conversely, primaquine's half-life, MRT, and volume distribution in the plasma were lower for NQ-Pq than for free primaquine. NE-Pq, on the other hand, accumulated more in the lungs but not in the liver. Galactose-coated primaquine nanochitosan formulation showed increased drug targeting to the liver compared to free primaquine and may represent a promising strategy for a more efficient and safer radical cure for vivax malaria.

In vivo photodynamic inactivation of Candida albicans using chloro-aluminum phthalocyanine.

This study evaluated the photoinactivation of Candida albicans in a murine model of oral candidiasis using chloro-aluminum phthalocyanine (ClAlP) encapsulated in cationic nanoemulsions (NE) and chloro-aluminum phthalocyanine (ClAlP) diluted in DMSO (DMSO) as photosensitizer (PS). Seventy-five 6-week-old female Swiss mice were immunosuppressed and inoculated with C. albicans to induce oral candidiasis. PDT was performed on the tongue by the application of the photosensitizers and LED light (100 J cm(-2) -660 nm). Twenty-four hours and 7 days after treatments, microbiological evaluation was carried out by recovering C. albicans from the tongue of animals (CFU ml(-1) ). Then, mice were sacrificed and the tongues were surgically removed for histological and biomolecular analysis of pro- and anti-inflammatory cytokines. Data were analyzed by ANOVA followed by Tukey's post hoc test. ClAlP-NE-mediated PDT reduced 2.26 log10 of C. albicans recovered from the tongue when compared with the control group (P-L-) (P < 0.05). PDT did not promote adverse effects on the tongue tissue. Seven days after treatment, all animals were completely healthy. In summary, PDT mediated by chloro-aluminum phthalocyanine entrapped in cationic nanoemulsions was effective in reducing C. albicans recovered from the oral lesions of immunocompromised mice.

Biocompatible magnetic microspheres for Use in PDT and hyperthermia.

Loaded microspheres with a silicon (IV) phthalocyanine derivative (NzPC) acting as a photosensitizer were prepared from polyhydroxybutyrate-co-valerate (PHBHV) and poly(ecaprolactone) (PCL) polymers using the emulsification solvent evaporation method (EE). The aim of our study was to prepare two systems of these biodegradable PHBHV/PCL microspheres. The first one containing only photosensitizer previously incorporated in the PHBHV and poly(ecaprolactone) (PCL) microspheres and the second one with the post magnetization of the DDS with magnetic nanoparticles. Magnetic fluid is successfully used for controlled incorporation of nanosized magnetic particles within the micron-sized template. This is the first time that we could get a successful pos incorporation of nanosized magnetic particles in a previously-prepared polymeric template. This procedure opens a great number of possibilities of post-functionalization of polymeric micro or nanoparticles with different bioactive materials. The NzPC release profile of the systems is ideal for PDT, the zeta potential and the size particle are stable upon aging in time. In vitro studies were evaluated using gingival fibroblastic cell line. The dark citotoxicity, the phototoxicity and the AC magnetic field assays of the as-prepared nanomagnetic composite were evaluated and the cellular viability analyzed by the classical test of MTT.

Photodynamic inactivation of biofilms formed by Candida spp., Trichosporon mucoides, and Kodamaea ohmeri by cationic nanoemulsion of zinc 2,9,16,23-tetrakis(phenylthio)-29H, 31H-phthalocyanine (ZnPc).

The biofilms formed by opportunistic yeasts serve as a persistent reservoir of infection and impair the treatment of fungal diseases. The aim of this study was to evaluate photodynamic inactivation (PDI) of biofilms formed by Candida spp. and the emerging pathogens Trichosporon mucoides and Kodamaea ohmeri by a cationic nanoemulsion of zinc 2,9,16,23-tetrakis(phenylthio)-29H,31H-phthalocyanine (ZnPc). Biofilms formed by yeasts after 48 h in the bottom of 96-well microtiter plates were treated with the photosensitizer (ZnPc) and a GaAlAs laser (26.3 J cm(-2)). The biofilm cells were scraped off the well wall, homogenized, and seeded onto Sabouraud dextrose agar plates that were then incubated at 37°C for 48 h. Efficient PDI of biofilms was verified by counting colony-forming units (CFU/ml), and the data were submitted to analysis of variance and the Tukey test (p < 0.05). All biofilms studied were susceptible to PDI with statistically significant differences. The strains of Candida genus were more resistant to PDI than emerging pathogens T. mucoides and K. ohmeri. A mean reduction of 0.45 log was achieved for Candida spp. biofilms, and a reduction of 0.85 and 0.84, were achieved for biofilms formed by T. mucoides and K. ohmeri, respectively. Therefore, PDI by treatment with nanostructured formulations cationic zinc 2,9,16,23- tetrakis (phenylthio)- 29H, 31H- phthalocyanine (ZnPc) and a laser reduced the number of cells in the biofilms formed by strains of C. albicans and non-Candida albicans as well the emerging pathogens T. mucoides and K. ohmeri.

Validated spectrophotometric and spectrofluorimetric methods for determination of chloroaluminum phthalocyanine in nanocarriers.

UV-VIS-Spectrophotometric and spectrofluorimetric methods have been developed and validated allowing the quantification of chloroaluminum phthalocyanine (CIAIPc) in nanocarriers. In order to validate the methods, the linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, and selectivity were examined according to USP 30 and ICH guidelines. Linearities range were found between 0.50-3.00 microg x mL(-1) (Y = 0.3829 X [CIAIPc, microg x mL(-1)] + 0.0126; r = 0.9992) for spectrophotometry, and 0.05-1.00 microg x mL(-1) (Y = 2.24 x 10(6) X [CIAIPc, microg x mL(-1)] + 9.74 x 10(4); r = 0.9978) for spectrofluorimetry. In addition, ANOVA and Lack-of-fit tests demonstrated that the regression equations were statistically significant (p<0.05), and the resulting linear model is fully adequate for both analytical methods. The LOD values were 0.09 and 0.01 microg x mL(-1), while the LOQ were 0.27 and 0.04 microg x mL(-1) for spectrophotometric and spectrofluorimetric methods, respectively. Repeatability and intermediate precision for proposed methods showed relative standard deviation (RSD) between 0.58% to 4.80%. The percent recovery ranged from 98.9% to 102.7% for spectrophotometric analyses and from 94.2% to 101.2% for spectrofluorimetry. No interferences from common excipients were detected and both methods were considered specific. Therefore, the methods are accurate, precise, specific, and reproducible and hence can be applied for quantification of CIAIPc in nanoemulsions (NE) and nanocapsules (NC).

Effect of berberine associated with photodynamic therapy in cell lines.

Cervical cancer is a serious worldwide health problem. In view of the potentially harmful effects of current conventional therapies, photodynamic therapy may be an option as it is a minimally invasive therapy and can promote selective cytotoxic activity for neoplastic cells in the target tissue., Berberine (BBR) as an isolated molecule is a natural compound that has antineoplastic properties and potential action as a photosensitizer agent. The purpose of this study was to evaluate the use of berberine as a photosensitizer in photodynamic therapy (PDT) protocols and observe the effects produced by this association in cervical carcinoma cells and in immortalized keratinocytes. Incubation with 2.5 µM berberine promoted less than 10 % of cellular death in both cell lines studied. In addition, by fluorescence microscopy, we demonstrated that berberine was internalized by the cells, and after a period of 48 h, it was still present in the intracellular environment preferentially localized in the cytoplasm. After photodynamic therapy using berberine as a photosensitizer and visible light activation at 447 (±10) nm, we observed a phototoxic effect, which resulted in 19.84 % cell viability for Caski cells and 47.22 % cell viability for HaCaT. Treatment with berberine associated with photodynamic therapy promoted an increase in the production of reactive species of oxygen (ROS) and caspase-3 activity, indicating a preferential cell death mechanism by caspase-dependent apoptosis. Therefore, we demonstrated that berberine is an efficient photosensitizer and that its association with photodynamic therapy may be a potential anticancer treatment strategy for cervical cancer.

Dynamic susceptibility investigation of maghemite nanoparticles incorporated in bovine serum albumin template.

Room-temperature measurements of the magnetic susceptibility of Bovine Serum Albumin-based nanocapsules (50 to 300 nm in size) loaded with different amounts of maghemite nanoparticles (7.6 nm average diameter) have been carried out in this study. The field (H) dependence of the imaginary peak susceptibility (fp) of the nanocomposite samples was investigated in the range of 0 to 4 kOe. From the analysis of the fp x H curves the concentration (N) dependence of the effective maghemite magnetocrystalline energy barrier (E) was obtained. Analysis of the E x N data was performed using a modified Mørup-Tronc [Phys. Rev. Lett. 72, 3278 (1994)] model, from which a huge contribution from the magnetocrystalline surface anisotropy was observed.

Evaluation of the binding properties of maghemite nanoparticle surface-coated with meso-2-3-dimercaptosuccinic acid to serum albumin.

In this study the interaction between magnetic nanoparticles (MNPs) surface-coated with meso-2,3-dimercaptosuccinic acid (DMSA) with both bovine serum albumin (BSA) and human serum albumin (HSA) was investigated. The binding of the MNP-DMSA was probed by the fluorescence quenching of the BSA and HSA tryptophan residue. Magnetic resonance and light microscopy analyses were carried out in in vivo tests using female Swiss mice. The binding constants (Kb) and the complex stoichiometries (n) indicate that MNP-DMSA/BSA and MNP-DMSA/HSA complexes have low association profiles. After five minutes following intravenous injection of MNP-DMSA into mice's blood stream we found the lung firstly target by the MNP-DMSA, followed by the liver in a latter stage. This finding suggests that the nanoparticle's DMSA-coating process probably hides the thiol group, through which albumin usually binds. This indicates that biocompatible MNP-DMSA is a very promising material system to be used as a drug delivery system (DDS), primarily for lung cancer treatment.

Analysis of mitochondrial activity related to cell death after PDT with AlPCS(4).

OBJECTIVE: The aim of this study was to report that photodynamic therapy (PDT) with mitochondria-associated chloroaluminum phthalocyanine tetrasulfonate (AlPcS(4)) leads to significant alterations in this organelle. BACKGROUND DATA: PDT is a viable treatment modality for a variety of tumors, as well as for some non-oncologic diseases. The procedure submits cells or tissue to a photosensitizing drug followed by light irradiation of appropriate wavelength, usually in the red area or close to infrared, and compatible with the drug absorption spectrum, inducing the apoptotic process. However, the precise mechanism of PDT-induced apoptosis is not well characterized. Several cellular organelles can be postulated as the target for PDT with different photosensitizers such as plasmatic membrane, nucleus, mitochondria, endoplasmic reticulum, Golgi complex, and others. The mitochondrion is the main target in PDT because it is the main organelle involved in apoptosis. One of the main agents is cytochrome c, a proapoptotic factor that preferentially links itself to the mitochondrial cardiolipin. METHODS: The photosensitizing effects of AlPcS(4) were studied in the mitochondria. Cells were irradiated with a diode laser (670 nm, energy density of 4.5 J/cm(2), and power density of 45 mW/cm(2)). RESULTS: The fluorescent analyses of the mitochondria were performed with MitoTracker and nonyl acridine orange (NAO), and electron microscopy demonstrated that PDT with AlPcS(4) leads to significant alterations in mitochondria, causing membrane potential loss, alteration in cardiolipin distribution and cell death. CONCLUSION: The labels with Mitotracker and NAO demonstrated mitochondrial migration to the perinuclear region, confirmed through electron microscopy, suggesting that intact mitochondria were solicited for possible DNA fragmentation.

Cell toxicity studies of albumin-based nanosized magnetic beads.

The aim of this study was to prepare bovine serum albumin-based beads containing maghemite nanoparticles incorporated via ionic magnetic fluid and to evaluate the cell toxicity of this biocompatible system using the J774-A1 cell line. Transmission electron micrographs obtained from the magnetic fluid sample were used to estimate the average particle diameter around 7.6 nm and diameter dispersion of 0.22. The BSA-based magnetic beads were prepared using the heat protein denaturation route. The nanoparticle concentration in the magnetic fluid sample used for the synthesis of the magnetic beads was in the range of 1.2 x 10(16) to 2.3 x 10(17) particle/ml. The methodology used to investigate the cell toxicity of the magnetic beads was the classical MTT assay. Our observation showed that the toxicity against the J774-A1 cell line depends upon the amount of magnetic material incorporated into the magnetic nanobeads and was found to be 14, 11, 9, 5, and 3% for 2.3 x 10(17), 1.2 x 10(17), 4.6 x 10(16), 2.3 x 10(16), and 1.2 x 10(16) particle/ml, respectively.

Comparison of photosensitized plasma membrane damage caused by singlet oxygen and free radicals.

The efficiency and selectivity of photosensitized damage to membrane functions may be influenced strongly by the identity of the initial reactive species formed by the photosensitizer. To test this possibility, a photosensitizer, rose bengal (RB), was used that resides in the plasma membrane and which generates singlet molecular oxygen (1O2*) upon excitation with visible light, and radicals plus 1O2* upon excitation with UV radiation. With this approach, 1O2* and radicals are formed at the same locations in the plasma membrane. The response of three plasma membrane functions, namely, proline transport, membrane potential, and membrane impermeability to charged dye molecules, was assessed. The efficiencies of the responses in the presence and absence of oxygen were compared per photon absorbed by RB at two wavelengths, 355 nm (UV excitation) and 532 nm (visible excitation). The efficiency of oxygen removal before irradiation was assessed by measuring the RB triplet lifetime. The three membrane functions were inhibited more efficiently at 355 nm than at 532 nm in the presence of oxygen indicating that the radicals are more effective at initiating damage to membrane components than 1O2*. The ratio of photosensitized effects at the two wavelengths in the presence of oxygen was the same for two membrane functions but not for the third suggesting that 1O2* and radicals initiate a common mechanistic pathway for damage to some membrane functions but not to others. Removing oxygen reduced the efficiency of 355 nm-induced photosensitization by factors of 1.4 to 7. The sensitivity of the three membrane functions to 1O2*-initiated damage varied over a factor of 50 whereas radical initiated damage only varied by a factor of 15. In summary, these results indicate that radicals and 1O2* formed at the same locations in the plasma membrane vary in their efficiency and specificity for membrane damage but may, in some cases, operate by a common secondary damage mechanism in the presence of oxygen.

Activity and in vivo tracking of Amphotericin B loaded PLGA nanoparticles.

The development of biocompatible polymeric nanoparticles has become an important strategy for optimizing the therapeutic efficacy of many classical drugs, as it may expand their activities, reduce their toxicity, increase their bioactivity and improve biodistribution. In this study, nanoparticles of Amphotericin B entrapped within poly (lactic-co-glycolic) acid and incorporated with dimercaptosuccinic acid (NANO-D-AMB) as a target molecule were evaluated for their physic-chemical characteristics, pharmacokinetics, biocompatibility and antifungal activity. We found high plasma concentrations of Amphotericin B upon treatment with NANO-D-AMB and a high uptake of nanoparticles in the lungs, liver and spleen. NANO-D-AMB exhibited antifungal efficacy against Paracoccidioides brasiliensis and induced much lower cytotoxicity levels compared to D-AMB formulation in vivo and in vitro. Together, these results confirm that NANO-D-AMB improves Amphotericin B delivery and suggest this delivery system as a potential alternative to the use of Amphotericin B sodium deoxycholate.

A vehicle for photodynamic therapy of skin cancer: influence of dimethylsulphoxide on 5-aminolevulinic acid in vitro cutaneous permeation and in vivo protoporphyrin IX accumulation determined by confocal microscopy.

Topical application of 5-aminolevulinic acid (5-ALA) followed by light irradiation is a new concept of photodynamic therapy (PDT) of skin cancers. 5-ALA is a prodrug that can be converted by the heme biosynthetic pathway into protoporphyrin IX, an effective photosensitizer. In the present work we propose the enhancement of 5-ALA-induced protoporphyrin IX accumulation by dimethylsulphoxide (DMSO) and ethylenediamine-tetraacetic acid disodium salt (EDTA). The presence of 20% DMSO (w/w) in oil-in-water emulsions increased the in vitro permeation of 5-ALA through hairless mouse skin. In vivo studies demonstrated a significant increase in the amount of protoporphyrin IX extracted from healthy hairless mouse skin after 3 h treatment with an oil-in-water emulsion containing 10% 5-ALA (w/w), 3% EDTA (w/w) and 20% DMSO (w/w). By confocal scanning laser microscopy imaging, an observed increase in red fluorescence, at 476 nm excitation and emission detected longer than 590 nm, in skin that had received this treatment, was attributed to protoporphyrin IX accumulation. Although no effect of EDTA on short-term protoporphyrin IX accumulation in skin was detected, this chelator could protect 5-ALA from decomposition during prolonged topical administration. The results obtained indicate that association of 5-ALA, EDTA and 20% DMSO may enhance the delivery of 5-ALA to the skin in the topical PDT.

Cubic phase gel as a drug delivery system for topical application of 5-ALA, its ester derivatives and m-THPC in photodynamic therapy (PDT).

The ability of the cubic liquid-crystalline phase to incorporate and control the release of drugs of varying size and polar characteristics makes it an interesting candidate as a drug delivery system. In the present study we investigated a new potential application of the cubic phase (monoolein/water; 70:30, w/w) to deliver pro-drugs and a photosensitizer for topical application in photodynamic therapy (PDT). Therefore the pro-drug 5-aminolevulinic acid (5-ALA, a PpIX precursor), its ester derivatives (hexylester, octylester and decylester), and the chlorine compound meso-tetra(hydroxyphenyl)chlorine (m-THPC) were incorporated into the cubic phase gel of monoolein/water and their physicochemical and spectroscopic properties were investigated at 37 degrees C. Drug stability was monitored for short and long periods of time. 5-ALA and its ester derivatives as non-fluorescent probes had their properties studied after chemical reaction leading to a fluorescent derivative. For all the compounds analyzed in this study the spectroscopic properties were clearly defined with potential photodynamic activity in the gel formulation. We are currently evaluating the potential of monoolein/water as a drug delivery system in the treatment of different cutaneous diseases and other PDT applications.

Photophysicals and photochemicals studies of zinc(II) phthalocyanine in long time circulation micelles for photodynamic therapy use.

Long time circulation systems, such as polymeric micelles, represent a growing area in biomedical research. These microparticles can be used in many biological systems to provide appropriate drug levels with a specific biodistribution. Long time circulation micelles (LTCM) were routinely prepared using PEG-5000-DSPE (polyethyleneglycol-5000-distearoil-phosphatidyl-ethanolamine) and zinc(II) phthalocyanine (ZnPc) as a photosensitizer and fluorescent probe. This compound belongs to a second generation of photoactive agents, mainly used in photodynamic therapy (PDT) of neoplasic tissues. Their high selectivity for tumoral target tissues as well as high phototoxicity based on singlet oxygen generation renders the utilization of these compounds feasible as an alternative therapy for cancer treatment. LTCM were characterized by classical spectroscopic techniques. Absorbance measurements indicated that the drug was s completely loaded into LTCM (epsilon = 2.41 x 10(5) cm(-1)). This was also verified by steady state and time-resolved fluorescence measurements. The lifetime profiles of ZnPc decay curves were fitted according to biexponential function (tau1 = 3.9 ns and tau2 = 15.5 ns) indicating different locations for ZnPc into LTCM. The time-resolved spectroscopy measurements for ZnPc triplet excited state lifetimes (tauT) were calculated from the kinetic analysis of transient decays at the absorption maximum (480 nm), by using laser flash photolysis technique. All the spectroscopy measurements performed allowed us to conclude that, ZnPc in LTCM is a promising drug delivery system (DDS) for PDT.

Photochemistry and photobiology of actinic erythema: defensive and reparative cutaneous mechanisms.

Sunlight is part of our everyday life and most people accept it as beneficial to our health. With the advance of our knowledge in cutaneous photochemistry, photobiology and photomedicine over the past four decades, the terrestrial solar radiation has become a concern of dermatologists and is considered to be a major damaging environmental factor for our skin. Most photobiological effects (e.g., sunburn, suntanning, local and systemic immunosuppression, photoaging or dermatoheliosis, skin cancer and precancer, etc.) are attributed to ultraviolet radiation (UVR) and more particularly to UVB radiation (290-320 nm). UVA radiation (320-400 nm) also plays an important role in the induction of erythema by the photosensitized generation of reactive oxygen species (singlet oxygen (1O2), superoxide (O2.-) and hydroxyl radicals (.OH)) that damage DNA and cellular membranes, and promote carcinogenesis and the changes associated with photoaging. Therefore, research efforts have been directed at a better photochemical and photobiological understanding of the so-called sunburn reaction, actinic or solar erythema. To survive the insults of actinic damage, the skin appears to have different intrinsic defensive mechanisms, among which antioxidants (enzymatic and non-enzymatic systems) play a pivotal role. In this paper, we will review the basic aspects of the action of UVR on the skin: a) photochemical reactions resulting from photon absorption by endogenous chromophores; b) the lipid peroxidation phenomenon, and c) intrinsic defensive cutaneous mechanisms (antioxidant systems). The last section will cover the inflammatory response including mediator release after cutaneous UVR exposure and adhesion molecule expression.

Nitric oxide release from the S-nitrosothiol zinc phthalocyanine complex by flash photolysis.

The photogeneration of nitric oxide (NO) using laser flash photolysis was investigated for S-nitroso-glutathione (GSNO) and S-nitroso-N-acetylcysteine (NacySNO) at pH 6.4 (PBS/HCl) and 7.4 (PBS). Irradiation of S-nitrosothiol with light (lambda = 355 nm followed by absorption spectroscopy) resulted in the homolytic decomposition of NacySNO and GSNO to generate radicals (GS and NacyS ) and NO. The release of NO from donor compounds measured with an ISO-Nometer apparatus was larger at pH 7.4 than pH 6.4. NacySNO was also incorporated into dipalmitoyl-phosphatidylcholine liposomes in the presence and absence of zinc phthalocyanine (ZnPC), a well-known photosensitizer useful for photodynamic therapy. Liposomes are usually used as carriers for hydrophobic compounds such as ZnPC. Inclusion of ZnPC resulted in a decrease in NO liberation in liposomal medium. However, there was a synergistic action of both photosensitizers and S-nitrosothiols resulting in the formation of other reactive species such as peroxynitrite, which is a potent oxidizing agent. These data show that NO release depends on pH and the medium, as well as on the laser energy applied to the system. Changes in the absorption spectrum were monitored as a function of light exposure.

The antioxidant action of Polypodium leucotomos extract and kojic acid: reactions with reactive oxygen species.

Two natural products Polypodium leucotomos extract (PL) and kojic acid (KA) were tested for their ability to scavenge reactive oxygen species (.OH,.O2-, H2O2, 1O2) in phosphate buffer. Hydroxyl radicals were generated by the Fenton reaction, and the rate constants of scavenging were 1.6 x 10(9) M-1 s-1 for KA and 1.0 x 10(9) M-1 s-1 for PL, similar to that of ethanol (1.4 x 10(9) M-1 s-1). With superoxide anions generated by the xanthine/hypoxanthine system, KA and PL (0.2-1.0 mg/ml) inhibited.O2-dependent reduction of nitroblue tetrazolium by up to 30 and 31%, respectively. In the detection of 1O2 by rose bengal irradiation, PL at 1.0 mg/ml quenched singlet oxygen by 43% relative to azide and KA by 36%. The present study demonstrates that PL showed an antioxidant effect, scavenging three of four reactive oxygen species tested here. Unlike KA, PL did not significantly scavenge hydrogen peroxide.

Photophysical studies of zinc phthalocyanine and chloroaluminum phthalocyanine incorporated into liposomes in the presence of additives.

The photophysical properties of zinc phthalocyanine (ZnPC) and chloroaluminum phthalocyanine (AlPHCl) incorporated into liposomes of dimyristoyl phosphatidylcholine in the presence and absence of additives such as cholesterol or cardiolipin were studied by time-resolved fluorescence, laser flash photolysis and steady-state techniques. The absorbance of the drugs changed linearly with drug concentration, at least up to 5.0 M in homogeneous and heterogeneous media, indicating that aggregation did not occur in these media within this concentration range. The incorporation of the drugs into liposomes increases the dimerization constant by one order of magnitude (for ZnPC, 3.6 x 10(4) to 1.0 x 10(5) M-1 and for AlPHCl, 3.7 x 10(4) to 1.5 x 10(5) M-1), but this feature dose does not rule out the use of this carrier, since the incorporation of these hydrophobic drugs into liposomes permits their systemic administration. Probe location in biological membranes and predominant positions of the phthalocyanines in liposomes were inferred on the basis of their fluorescence and triplet state properties. Both phthalocyanines are preferentially distributed in the internal regions of the liposome bilayer. The additives affect the distribution of these drugs within the liposomes, a fact that controls their delivery when both are used in a biological medium, retarding their release. The addition of the additives to the liposomes increases the internalization of phthalocyanines. The interaction of the drugs with a plasma protein, bovine serum albumin, was examined quantitatively by the fluorescence technique. The results show that when the drugs were incorporated into small unilamellar liposomes, the association with albumin was enhanced when compared with organic media, a fact that should increase the selectivity of tumor targeting by these phthalocyanines (for ZnPC, 0.71 x 10(6) to 1.30 x 10(7) M-1 and for AlPHCl, 4.86 x 10(7) to 3.10 x 10(8) M-1).

Photocytotoxicity of a 5-nitrofuran-ethenyl-quinoline antiseptic (Quinifuryl) to P388 mouse leukemia cells.

Quinifuryl (MW 449.52), 2-(5'-nitro-2'-furanyl)ethenyl-4-[N-[4'-(N,N-diethylamino)-1'-methylbutyl]carbamoyl] quinoline, is a water soluble representative of a family of 5-nitrofuran-ethenyl-quinoline drugs which has been shown to be highly toxic to various lines of transformed cells in the dark. In the present study, the toxicity of Quinifuryl to P388 mouse leukemia cells was compared in the dark and under illumination with visible light (390-500 nm). Illumination of water solutions of Quinifuryl (at concentrations ranging from 0.09 to 9.0 microg/ml) in the presence of P388 cells resulted in its photodecomposition and was accompanied by elevated cytotoxicity. A significant capacity to kill P388 cells was detected at a drug concentration as low as 0.09 microg/ml. The toxic effect detected at this drug concentration under illumination exceeded the effect observed in the dark by more than three times. Moreover, the general toxic effect of Quinifuryl, which included cell proliferation arrest, was nearly 100%. Both dose- and time-dependent toxic effects were measured under illumination. The LC50 value of Quinifuryl during incubation with P388 cells was approximately 0.45 microg/ml under illumination for 60 min and >12 microg/ml in the dark. We have demonstrated that the final products of the Quinifuryl photolysis are not toxic, which means that the short-lived intermediates of Quinifuryl photodecomposition are responsible for the phototoxicity of this compound. The data obtained in the present study are the first to indicate photocytotoxicity of a nitroheterocyclic compound and demonstrate the possibility of its application as a photosensitizer drug for photochemotherapy.