ABSTRACT
INTRODUCTION AND OBJECTIVE: Photodynamic therapy (PDT) is a therapeutic option for low-risk basal cell carcinoma (BCC). The aim of the study was to assess the efficacy of topical PDT in the treatment of superficial BCC (sBCC) using two different photosensitizers: aminolevulinic acid hydrochloride (ALA-HCl) in a gel formulation with a lipid nanoemulsion (ALA-HCl in gel) and ALA methyl ester hydrochloride (MAL-HCl) in a cream formulation (MAL-HCl in cream). MATERIAL AND METHODS: 21 patients were treated twice with a one week interval between treatments. The formulations were applied onto lesions: 10 patients were treated with MAL-HCl in cream, and 11 with ALA-HCl in gel. After three hours of incubation and removing the preparations, fluorescence was assessed. The skin areas were then irradiated with red light 630 ± 5 nm. RESULTS: At the follow-up visit 12 weeks after the second treatment, complete clinical remission was found in 82% after ALA-HCl in gel and in 80% after MAL-HCl in cream. An excellent cosmetic result was found in 96% of patients after MALHCl in cream and in 100% after ALA-HCl in gel. Faster skin healing and less post-inflammatory hyperpigmentation during follow-up visits was observed after treatment with ALA-HCl in gel. CONCLUSIONS: Both formulations - ALA-HCl in gel and MAL-HCl in cream - were highly effective photosensitisers for PDT. The advantage of ALA-HCl in a gel formulation with a lipid nanoemulsion was faster skin healing, resulting in better cosmetic results.
Subject(s)
Carcinoma, Basal Cell , Photochemotherapy , Skin Neoplasms , Humans , Skin Neoplasms/drug therapy , Skin Neoplasms/etiology , Skin Neoplasms/pathology , Photochemotherapy/methods , Treatment Outcome , Carcinoma, Basal Cell/drug therapy , Carcinoma, Basal Cell/etiology , Carcinoma, Basal Cell/pathology , Aminolevulinic Acid/therapeutic use , Aminolevulinic Acid/toxicity , Pathologic Complete Response , LipidsABSTRACT
Yinzhihuang oral liquid (YZH) is a traditional Chinese medicine that has been widely used in Asia to prevent and treat neonatal hyperbilirubinemia, but the published preclinical studies on its anti-hyperbilirubinemia effect are conducted in adult animals, partly due to the lack of preclinical neonatal hyperbilirubinemia animal models. In the present study, we tested six reagents to induce hyperbilirubinemia in neonatal rats, and established two appropriate neonatal hyperbilirubinemia rat models by subcutaneous injection of δ-Aminolevulinic acid (ALA, 200â mg/kg) or novobiocin (NOVO, 200â mg/kg). Oral treatment of YZH (80, 160 and 320â mg/kg) significantly decreased serum conjugated bilirubin levels in ALA-treated neonatal rats and serum unconjugated bilirubin levels in NOVO-treated neonatal rats, respectively. Additionally, pre-treatment of YZH also prevented the increase of serum bilirubin levels in both ALA- and NOVO-treated rats. Mechanistically, YZH significantly up-regulated the mRNA expression of genes involved in hepatic bilirubin disposition (organic anion-transporting polypeptide 1b2, Oatp1b2; multidrug resistance-associated proteinâ 2, Mrp2) and bilirubin conjugation (UDP-glucuronosyltransferase 1a1, Ugt1a1). Additionally, YZH up-regulated the mRNA expression of cytochrome P450 1A1 (Cyp1a1), the target gene of aryl hydrocarbon receptor (AhR), and increased the nuclear protein levels of AhR in livers of neonatal rats. YZH and its two active ingredients, namely baicalin (BCL) and 4'-hydroxyacetophenone (4-HT), up-regulated the mRNA expression of AhR target genes (CYP1A1 and UGT1A1) and increased nuclear protein levels of AhR in HepG2 cells. In conclusion, the present study provides two neonatal hyperbilirubinemia animal models and evaluates the anti-hyperbilirubinemia effect and mechanisms of YZH in neonatal animals.
Subject(s)
Drugs, Chinese Herbal/chemistry , Administration, Oral , Aminolevulinic Acid/toxicity , Animals , Animals, Newborn , Bilirubin/blood , Cytochrome P-450 CYP1A1/genetics , Cytochrome P-450 CYP1A1/metabolism , Drugs, Chinese Herbal/pharmacology , Drugs, Chinese Herbal/therapeutic use , Hep G2 Cells , Humans , Hyperbilirubinemia/chemically induced , Hyperbilirubinemia/drug therapy , Hyperbilirubinemia/pathology , Liver/drug effects , Liver/metabolism , Liver/pathology , Medicine, Chinese Traditional , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins/genetics , Multidrug Resistance-Associated Proteins/metabolism , Novobiocin/toxicity , RNA Interference , RNA, Small Interfering/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Aryl Hydrocarbon/antagonists & inhibitors , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism , Up-Regulation/drug effectsABSTRACT
BACKGROUND: 5-Aminolevulinic acid (5-ALA), a fluorescent contrast agent, has been used for tumor paint and photodynamic therapy (PDT) for various tumors, but its use with soft tissue sarcomas is not well documented. Myxofibrosarcoma, a subtype of soft tissue sarcoma with a high local recurrence rate, may benefit from similar types of treatment. The purpose of this study was to analyze the effects of 5-ALA tumor paint and PDT on a myxofibrosarcoma cell line. METHODS: Tumor paint was assessed by exposing micromass pellets of human adipose-derived stromal (ADS) cells or myxofibrosarcoma (MUG-Myx1) cells to 5-ALA. Cell pellets were then visualized using a microscope at established excitation and emission wavelengths. Corrected total cell fluorescence was calculated per accepted protocols. Photodynamic therapy was similarly assessed by exposing ADS and MUG-Myx1 cells to 5-ALA, with subsequent analysis via flow cytometry and real-time confocal microscopy. RESULTS: The use of 5-ALA tumor paint led to a selective fluorescence in MUG-Myx1 cells. Findings were confirmed by flow cytometry. Interestingly, flow cytometry results showed progressive selective cell death with increasing 5-ALA exposure as a result of the PDT effect. PDT was further confirmed using confocal microscopy, which revealed progressive cellular bubble formation consistent with advancing stages of cell death-a finding that was not seen in control ADS cells. CONCLUSIONS: 5-ALA tumor paint and PDT were successfully used on a human myxofibrosarcoma cell line (MUG-Myx1). Results from this study showed both selective fluorescent tagging and selective cytotoxicity of 5-ALA toward malignant myxofibrosarcoma cells, while sparing benign adipose control cells. This finding was further confirmed in a dramatic time-lapse video, visually confirming active, targeted cell death. 5-ALA's two-pronged application of selective tumor identification and cytotoxicity may transform surgical and medical approaches for treating soft tissue sarcomas.
Subject(s)
Aminolevulinic Acid/toxicity , Contrast Media/toxicity , Fibroma/therapy , Fibrosarcoma/therapy , Photochemotherapy/methods , Aminolevulinic Acid/analysis , Aminolevulinic Acid/therapeutic use , Cell Line, Tumor , Contrast Media/analysis , Contrast Media/therapeutic use , Fibroma/diagnosis , Fibrosarcoma/diagnosis , Humans , Microscopy, Confocal/methodsABSTRACT
Metronomic photodynamic therapy (mPDT) was developed to improve tumor-specific responses through cell death by apoptosis. We developed an mPDT suppository kit including ALA and LED suppositories and analyzed its killing effect on rectal tumors in rabbits. METHODS: The ALA (10â¯wt%) suppository was prepared using ALA powder, type 36 semi-synthetic fatty acid glyceride, and azone. The LED suppository was constructed by encapsulating LED units and a circuit in transparent epoxy resin. VX2 cells were injected into the rectal submucosa of rabbits to establish a carcinoma model in situ. The ALA suppository was inserted into the rectal cavity for 30â¯min of uptake and activated for 1â¯h by the LED suppository at a power density of 20â¯mW/cm2. The mPDT process was repeated three times once a day. MRI was used to monitor tumor growth, histopathology and TUNEL staining were performed at 14â¯days after mPDT. RESULTS: The overall response rate was 60% in the mPDT group using the kit in which the tumor size was decreased up to about 50% at 7â¯days post-mPDT and almost eliminated at 14â¯days. HE staining showed that only 6.16% of the tumor tissue remained after mPDT treatment. TUNEL detection showed that the apoptosis rate was 18.9%. CONCLUSION: We verified the killing effect of the mPDT suppository kit on rectal tumors in rabbits based on mPDT that induced tumor cell apoptosis.
Subject(s)
Apoptosis/drug effects , Photosensitizing Agents/toxicity , Suppositories/chemistry , Aminolevulinic Acid/toxicity , Animals , Apoptosis/radiation effects , Cell Line, Tumor , Light , Magnetic Resonance Imaging , Photochemotherapy , Photosensitizing Agents/therapeutic use , Rabbits , Rectal Neoplasms/diagnostic imaging , Rectal Neoplasms/drug therapy , Rectal Neoplasms/pathology , Transplantation, HomologousABSTRACT
An innovative microcarrier based on a carboxy-enriched and branched polylactic acid derivative was developed to enhance the in vitro phototoxicity of the photosensitizer and prodrug 5-aminolevulinic. Microparticles, prepared by double emulsion technique and loaded with the prodrug were carefully characterized and the effect of the polymer structure on the chemical, physical and biological properties of the final product was evaluated. Results showed that microparticles have a spherical shape and ability to allocate up to 30⯵g of the photosensitizer per mg of carrier despite their difference in solubility. Release studies performed in various simulated physiological conditions demonstrate the influence of the branched structure and the presence of the additional carboxylic groups on the release rate and the possibility to modulate it. In vitro assays conducted on human epithelial adenocarcinoma cells proved the not cytotoxicity of the carriers in a wide range of concentrations. The hemocompatibility and surface proteins adsorption were evaluated at different microparticles concentrations to evaluate the safety and estimate the possible microparticles residential time in the bloodstream. The advantages, of loading 5-aminolevulinic acid in the prepared carrier has been deeply described in terms of enhanced phototoxicity, compared to the free 5-aminolevulinic acid formulation after irradiation with light at 635â¯nm. The obtained results demonstrate the advantages of the prepared derivative compared to the linear polylactide for future application in photodynamic therapy based on the photosensitizer 5-aminolevulinic acid.
Subject(s)
Aminolevulinic Acid/chemistry , Photosensitizing Agents/chemistry , Polyesters/chemistry , Aminolevulinic Acid/metabolism , Aminolevulinic Acid/toxicity , Cell Line, Tumor , Cell Survival/drug effects , Drug Carriers/chemistry , Drug Liberation , Erythrocytes/cytology , Erythrocytes/drug effects , Erythrocytes/metabolism , HeLa Cells , Hemolysis/drug effects , Humans , Hydrogen-Ion Concentration , Light , Photosensitizing Agents/toxicity , SolubilityABSTRACT
Chromoblastomycosis (CBM), one of the neglected tropical diseases, is hard to cure and easy to be recurrent. Many studies suggest that macrophage is involved in the pathogenesis of chromoblastomycosis and the fungicidal effect of 5-Aminolaevulinic Acid-Based Photodynamic Therapy (ALA-PDT) against F. monophora (one of the main causative agent of chromoblastomycosis) has shown great promise. However, the fungicidal ability of ALA-PDT to F. monophora is still controversial and the molecular mechanism and immune mechanism of ALA-PDT against F. monophora remains poorly documented. In the present work, ALA (5-Aminolaevulinic Acid) was employed as photosensitizer and a LED device was served as light source to investigate photodynamic effect on F. monophora conidia under different ALA-PDT conditions in a direct way. RAW264.7 was stimulated by conidia treated with ALA-PDT to study the photodynamic effect on F. monophora conidia in an indirect way. It was observed that ALA-PDT can inactivate F. monophora conidia directly in a concentration-dependent and dose-dependent manner. RAW264.7 was activated indirectly by photodynamically treated conidia. ALA-PDT can enhance the fungicidal ability of RAW264.7 and protect it from Infection-induced apoptosis in an indirect way. ROS generated by photodynamic treated conidia is associated with mitochondrial-related apoptosis in RAW264.7.The results of this investigation demonstrated that ALA-PDT inactivate F. monophora through two way: directly killing F. monophora conidia through ROS-dependent Oxidative damage; activating RAW264.7 in an indirect way.
Subject(s)
Aminolevulinic Acid/pharmacology , Ascomycota/physiology , Photosensitizing Agents/pharmacology , Spores, Fungal/drug effects , Aminolevulinic Acid/therapeutic use , Aminolevulinic Acid/toxicity , Animals , Apoptosis/drug effects , Chromoblastomycosis/drug therapy , Light , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Photochemotherapy , Photosensitizing Agents/therapeutic use , Photosensitizing Agents/toxicity , RAW 264.7 Cells , Reactive Oxygen Species/metabolismABSTRACT
5-aminolevulinic acid mediated PDT (5-ALA-PDT) is an approved therapeutic procedure for treating carcinomas of the cervix. However, when employed as a monotherapy, 5-ALA-PDT could not produce satisfactory results toward large and deep tumors. Therefore, developing a method to improve the efficacy of 5-ALA-PDT becomes important. In this study, we demonstrate an enhanced antitumor effect of 5-ALA-PDT by the modulation of mitochondrial morphology. The mitochondria in the cells were regulated into tubular mitochondria or fragmented mitochondria through over expression of Drp1 or Mfn2. Then these cells were treated with identical dose of 5-ALA-PDT. Our results suggest that HeLa cells predominantly containing fragmented mitochondria were more sensitive to 5-ALA-PDT than the cells predominantly containing tubular mitochondria. The morphology of mitochondria changed as the cell cycle progressed, with tubular mitochondria predominantly exhibited in the S phase and uniformly fragmented mitochondria predominantly displayed in the M phase. Paclitaxel significantly increased the population of M-phase cells, while 5-fluorouracil significantly increased the population of S-phase cells in xenograft tumors. Furthermore, low-dose paclitaxel significantly increased the antitumor effects of PDT. However, 5-fluorouracil didn't improve the antitumor effects of PDT. These results demonstrated an enhanced antitumor effect of 5-ALA-PDT from the modulation of mitochondrial morphology. We anticipate that our results will provide an insight for selecting potential chemotherapeutic agents to combine with PDT for tumor treatment.
Subject(s)
Aminolevulinic Acid/toxicity , Antineoplastic Agents/toxicity , Apoptosis/drug effects , Mitochondria/chemistry , Photosensitizing Agents/toxicity , Aminolevulinic Acid/chemistry , Aminolevulinic Acid/therapeutic use , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/therapeutic use , Cell Cycle Checkpoints/drug effects , Cell Cycle Checkpoints/radiation effects , Dynamins , Fluorouracil/therapeutic use , Fluorouracil/toxicity , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , HeLa Cells , Humans , Immunohistochemistry , Membrane Potential, Mitochondrial/drug effects , Mice, Inbred BALB C , Mice, Nude , Microscopy, Fluorescence , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondria/radiation effects , Mitochondrial Proteins/genetics , Mitochondrial Proteins/metabolism , Neoplasms/drug therapy , Neoplasms/mortality , Neoplasms/pathology , Paclitaxel/therapeutic use , Paclitaxel/toxicity , Photochemotherapy , Photosensitizing Agents/chemistry , Photosensitizing Agents/therapeutic use , Plasmids/genetics , Plasmids/metabolism , Rats , Reactive Oxygen Species/metabolism , Survival Rate , Transplantation, HeterologousABSTRACT
Polysaccharides based nanocomplexes have been developed for encapsulation, controlled delivery and to enhance the phototoxicity of the photosensitizer 5-aminolevulinic acid for application in photodynamic therapy. The nanocomplexes were prepared by coacervation in a solvent free environment using chitosan as polycation while alginic and polygalacturonic acid as polyanions. The complexes showed average dimension in the range 90-120nm, good stability in simulated physiological media and high drug encapsulation efficiency, up to 800µg per mg of carrier. Release studies demonstrate the possibility to tune the overall release rate and the intensity of the initial burst by changing the external pH. Cytotoxicity and photocytotoxicity tests confirmed the not toxicity of the used polysaccharides. Cell viability results confirmed the improvement of 5-aminolevulinic acid phototoxicity when loaded into the carrier compared to the free form. No effect of the irradiation on the nanocomplexes structure and on the release kinetics of the drug was observed. The results demonstrate that the prepared formulations have suitable properties for future application in photodynamic therapy and to ameliorate the therapeutic efficacy and overcome the side-effects related to the use of the photosensitizer 5-aminolevulinic acid.
Subject(s)
Aminolevulinic Acid/chemistry , Chitosan/chemistry , Drug Carriers/chemistry , Nanoparticles/chemistry , Photosensitizing Agents/chemistry , Aminolevulinic Acid/toxicity , Cell Survival/drug effects , Cell Survival/radiation effects , Drug Liberation , HeLa Cells , Humans , Hydrogen-Ion Concentration , Light , Particle Size , Photosensitizing Agents/toxicity , TemperatureABSTRACT
Selection of effective herbicides to control weeds has been one of the major objectives of scientists. This study determines the differential tolerance or susceptibility of crickweed (Malachium aquaticum L.) to various concentration combinations of 5-aminolevulinic acid (ALA) (1, 10 and 100mg/L) and propyl 4-(2-(4,6-dimethoxypyrimidin-2-yloxy)benzylamino)benzoate (ZJ0273) (100, 200, and 500mg/L). ALA was applied as pre- and post-treatment alone or in combination with ZJ0273. Results showed that ZJ0273 stress alone imposed negative effects on M. aquaticum seedling's growth, net photosynthetic rates and SPAD values, and the rate of decline was consistently increased with the increase in ZJ0273 concentration. The ZJ0273 treatment showed a gradual decrease in the activities of antioxidant enzymes peroxidase (POD), superoxide dismutase (SOD), and ascorbate peroxidase (APX), and increase in the accumulation of malondialdehyde (MDA). Changes in chloroplast swelling, increased number of plastoglobuli, disruption of thylakoid, disintegrated mitochondria and turbid nucleoplasm were noticed. Moreover, SDS-PAGE analysis of total proteins revealed that herbicide stress in the leaves was associated with the decrease or disappearance of some protein bands. Further, two-dimensional gel electrophoresis (2-DE) results showed that proteins in different spots were classified into three types for M. aquaticum. These results indicate that the combined treatment of ALA and ZJ0273 synergizes the herbicide toxicity which is different from its independent effects on M. aquaticum and thus, could improve weed control efficacy.
Subject(s)
Aminolevulinic Acid/toxicity , Herbicides/toxicity , Plant Weeds/drug effects , Ascorbate Peroxidases/metabolism , Catalase/metabolism , Drug Synergism , Malondialdehyde/metabolism , Photosynthesis/drug effects , Plant Leaves/drug effects , Plant Leaves/enzymology , Plant Leaves/metabolism , Plant Leaves/ultrastructure , Plant Proteins/metabolism , Plant Weeds/enzymology , Plant Weeds/metabolism , Plant Weeds/ultrastructure , Superoxide Dismutase/metabolismABSTRACT
This paper focuses on the molecular mechanism of deregulated porphyrin biosynthesis in rice plants under photodynamic stress imposed by an exogenous supply of 5-aminolevulinic acid (ALA) and oxyfluorfen (OF). Plants treated with 5 mM ALA or 50 µM OF exhibited differential herbicidal symptoms as characterized by white and brown necrosis, respectively, with substantial increases in cellular leakage and malondialdehyde production. Protoporphyrin IX accumulated to higher levels after 1 day of ALA and OF treatment, whereas it decreased to the control level after 2 days of ALA treatment. Plants responded to OF by greatly decreasing the levels of Mg-protoporphyrin IX (MgProto IX), MgProto IX methyl ester, and protochlorophyllide to levels lower than control, whereas their levels drastically increased 1 day after ALA treatment and then disappeared 2 days after the treatment. Enzyme activity and transcript levels of HEMA1, GSA and ALAD for ALA synthesis greatly decreased in ALA- and OF-treated plants. Transcript levels of PPO1, CHLH, CHLI, and PORB genes involving Mg-porphyrin synthesis continuously decreased in ALA- and OF-treated plants, with greater decreases in ALA-treated plants. By contrast, up-regulation of FC2 and HO2 genes in Fe-porphyrin branch was noticeable in ALA and OF-treated plants 1 day and 2 days after the treatments, respectively. Decreased transcript levels of nuclear-encoded genes Lhcb1, Lhcb6, and RbcS were accompanied by disappearance of MgProto IX in ALA- and OF-treated plants after 2 days of the treatments. Under photodynamic stress imposed by ALA and OF, tight control of porphyrin biosynthesis prevents accumulation of toxic metabolic intermediates not only by down-regulation of their biosynthesis but also by photodynamic degradation. The up-regulation of FC2 and HO2 also appears to compensate for the photodynamic stress-induced damage.
Subject(s)
Aminolevulinic Acid/toxicity , Halogenated Diphenyl Ethers/toxicity , Herbicides/toxicity , Oryza/drug effects , Photosensitizing Agents/toxicity , Porphyrins/biosynthesis , Gene Expression Regulation, Plant/drug effects , Light , Oryza/genetics , Oryza/metabolism , Oryza/radiation effects , Photosynthesis/genetics , Porphyrins/genetics , Stress, Physiological/geneticsABSTRACT
Two-photon excitation of a sensitizer with a focused laser beam was used to create a spatially-localized subcellular population of reactive oxygen species, ROS, in single HeLa cells. The sensitizer used was protoporphyrin IX, PpIX, endogenously derived from 5-aminolevulinic acid delivered to the cells. Although we infer that singlet oxygen, O2(a(1)Δg), is one ROS produced upon irradiation of PpIX under these conditions, it is possible that the superoxide ion, O2(-Ë), may also play a role in this system. With a "high" dose of PpIX-sensitized ROS, the expected death of the cell was observed. However, under "low dose" conditions, clear signs of cell proliferation were observed. The present results facilitate studies of ROS-mediated signalling in imaging-based single cell experiments.
Subject(s)
Reactive Oxygen Species/metabolism , Singlet Oxygen/metabolism , Aminolevulinic Acid/chemistry , Aminolevulinic Acid/therapeutic use , Aminolevulinic Acid/toxicity , Apoptosis/drug effects , Apoptosis/radiation effects , Cell Proliferation/drug effects , Cell Proliferation/radiation effects , HeLa Cells , Humans , Lasers , Neoplasms/drug therapy , Photochemotherapy , Photosensitizing Agents/chemistry , Photosensitizing Agents/therapeutic use , Photosensitizing Agents/toxicity , Protoporphyrins/chemistry , Protoporphyrins/therapeutic use , Protoporphyrins/toxicityABSTRACT
Photodynamic Therapy (PDT) is an anticancer treatment based on photosensitisation of malignant cells. The precursor of the photosensitiser Protoporphyrin IX, 5-aminolevulinic acid (ALA), has been used for PDT of bladder cancer. Silybin is a flavonoid extracted from Silybum marianum, and it has been reported to increase the efficacy of several anticancer treatments. In the present work, we evaluated the cytotoxicity of the combination of ALA-PDT and silybin in the T24 and MB49 bladder cancer cell lines. MB49 cells were more sensitive to PDT damage, which was correlated with a higher Protoporphyrin IX production from ALA. Employing lethal light doses 50% (LD50) and 75% (LD75) and additional silybin treatment, there was a further increase of toxicity driven by PDT in both cell lines. Using the Chou-Talalay model for drug combination derived from the mass-action law principle, it was possible to identify the effect of the combination as synergic when using LD75, whilst the use of LD50 led to an additive effect on MB49 cells. On the other hand, the drug combination turned out to be nearly additive on T24 cells. Apoptotic cell death is involved both in silybin and PDT cytotoxicity in the MB49 line but there is no apparent correlation with the additive or synergic effect observed on cell viability. On the other hand, we found an enhancement of the PDT-driven impairment of cell migration on both cell lines as a consequence of silybin treatment. Overall, our results suggest that the combination of silybin and ALA-PDT would increase PDT outcome, leading to additive or synergistic effects and possibly impairing the occurrence of metastases.
Subject(s)
Apoptosis/drug effects , Photosensitizing Agents/toxicity , Silymarin/pharmacology , Aminolevulinic Acid/chemistry , Aminolevulinic Acid/pharmacology , Aminolevulinic Acid/toxicity , Cell Line, Tumor , Cell Movement/drug effects , Drug Synergism , Drug Therapy, Combination , Flavonoids/chemistry , Flavonoids/pharmacology , Flavonoids/therapeutic use , Humans , Light , Silybum marianum/chemistry , Silybum marianum/metabolism , Photochemotherapy , Photosensitizing Agents/chemistry , Photosensitizing Agents/therapeutic use , Silybin , Silymarin/chemistry , Silymarin/therapeutic use , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/pathologyABSTRACT
We present a novel high-throughput microfluidic platform that enables the evaluation of the anticancer efficacy of photodynamic therapy (PDT) drugs over multiple microenvironmental factors. PDT is uniquely complex, originating from its dependence on three separate but essential elements: drug (also called photosensitizer), oxygen, and light. Thus, obtaining a reliable evaluation of PDT efficacy is highly challenging, requiring considerable effort and time to evaluate all three interdependent parameters. In this paper, we report a high-throughput efficacy screening platform that we implemented by developing microfluidic components that individually control basic PDT elements (photosensitizer concentrations, oxygen levels, and light fluence) and then integrating them into a single triple-layer device. The integrated microfluidic chip consists of an array of small compartments, each corresponding to a specific combination of these three variables. This allows for more than 1000 different conditions being tested in parallel. Cancer cells are cultured within the device, exposed to different PDT conditions, and then monitored for their viability using live/dead fluorescence staining. The entire screening assay takes only 1 hour, and the collected PDT outcomes (cell viability) for combinatorial screening are analysed and reported as traditional dose-response curves or 3D bubble charts using custom software. As a proof of concept, methylene blue is adopted as a photosensitizer and its drug efficacy on C6 glioma cells has been successfully evaluated for a total of 324 PDT conditions using the fabricated chip. This platform can facilitate not only the development of new photosensitizers but also the optimization of current PDT protocols.
Subject(s)
Microfluidic Analytical Techniques/methods , Oxygen/analysis , Photosensitizing Agents/analysis , Aminolevulinic Acid/analysis , Aminolevulinic Acid/therapeutic use , Aminolevulinic Acid/toxicity , Brain Neoplasms/drug therapy , Cell Line, Tumor , Cell Survival/drug effects , Glioma/drug therapy , Humans , Light , Microfluidic Analytical Techniques/instrumentation , Photochemotherapy , Photosensitizing Agents/pharmacology , Photosensitizing Agents/therapeutic useABSTRACT
BACKGROUND: 5-Aminolevulinic acid (ALA) is a prodrug for topical photodynamic therapy. The effectiveness of topical ALA can be limited by its bioavailability. The aim of this study was to develop a novel ALA delivery approach using poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). METHODS: A modified double emulsion solvent evaporation method was used to prepare ALA loaded PLGA NPs (ALA PLGA NPs). The characteristics, uptake, protoporphyrin IX fluorescence kinetics, and cytotoxicity of ALA PLGA NPs toward a human skin squamous cell carcinoma cell line were examined. RESULTS: The mean particle size of spherical ALA PLGA NPs was 65.6 nm±26 nm with a polydispersity index of 0.62. The encapsulation efficiency was 65.8%±7.2% and ALA loading capacity was 0.62%±0.27%. When ALA was dispersed in PLGA NPs, it turned into an amorphous phase. ALA PLGA NPs could be taken up by squamous cell carcinoma cells and localized in the cytoplasm. The protoporphyrin IX fluorescence kinetics and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay showed that ALA PLGA NPs were more effective than free ALA of the same concentration. CONCLUSION: PLGA NPs provide a promising ALA delivery strategy for topical ALA-photodynamic therapy of skin squamous cell carcinoma.
Subject(s)
Aminolevulinic Acid/chemistry , Lactic Acid/chemistry , Nanoparticles/chemistry , Photosensitizing Agents/chemistry , Polyglycolic Acid/chemistry , Aminolevulinic Acid/pharmacokinetics , Aminolevulinic Acid/toxicity , Analysis of Variance , Calorimetry, Differential Scanning , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/radiation effects , Humans , Kinetics , Lactic Acid/pharmacokinetics , Lactic Acid/toxicity , Microscopy, Electron, Transmission , Nanoparticles/toxicity , Particle Size , Photochemotherapy/methods , Photosensitizing Agents/pharmacokinetics , Photosensitizing Agents/toxicity , Polyglycolic Acid/pharmacokinetics , Polyglycolic Acid/toxicity , Polylactic Acid-Polyglycolic Acid CopolymerABSTRACT
Accumulation of protoporphyrin IX (PpIX) in malignant cells is the basis of 5-aminolevulinic acid (ALA)-mediated photodynamic therapy. We studied the expression of proteins that possibly affect ALA-mediated PpIX accumulation, namely oligopeptide transporter-1 and -2, ferrochelatase and ATP-binding cassette transporter G2 (ABCG2), in several tumor cell lines. Among these proteins, only ABCG2 correlated negatively with ALA-mediated PpIX accumulation. Both a subcellular fractionation study and confocal laser microscopic analysis revealed that ABCG2 was distributed not only in the plasma membrane but also intracellular organelles, including mitochondria. In addition, mitochondrial ABCG2 regulated the content of ALA-mediated PpIX in mitochondria, and Ko143, a specific inhibitor of ABCG2, enhanced mitochondrial PpIX accumulation. To clarify the possible roles of mitochondrial ABCG2, we characterized stably transfected-HEK (ST-HEK) cells overexpressing ABCG2. In these ST-HEK cells, functionally active ABCG2 was detected in mitochondria, and treatment with Ko143 increased ALA-mediated mitochondrial PpIX accumulation. Moreover, the mitochondria isolated from ST-HEK cells exported doxorubicin probably through ABCG2, because the export of doxorubicin was inhibited by Ko143. The susceptibility of ABCG2 distributed in mitochondria to proteinase K, endoglycosidase H and peptide-N-glycosidase F suggested that ABCG2 in mitochondrial fraction is modified by N-glycans and trafficked through the endoplasmic reticulum and Golgi apparatus and finally localizes within the mitochondria. Thus, it was found that ABCG2 distributed in mitochondria is a functional transporter and that the mitochondrial ABCG2 regulates ALA-mediated PpIX level through PpIX export from mitochondria to the cytosol.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Aminolevulinic Acid/metabolism , Mitochondria/metabolism , Neoplasm Proteins/metabolism , Protoporphyrins/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Adenosine/analogs & derivatives , Adenosine/pharmacology , Aminolevulinic Acid/pharmacology , Aminolevulinic Acid/toxicity , Biological Transport , Cell Line, Tumor , Diketopiperazines , Doxorubicin/metabolism , Endoplasmic Reticulum/metabolism , Ferrochelatase/metabolism , Gene Expression , Glycosylation , Golgi Apparatus/metabolism , HEK293 Cells , Heterocyclic Compounds, 4 or More Rings , Humans , Neoplasm Proteins/genetics , Peptide Transporter 1 , Protein Transport , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Symporters/metabolism , U937 CellsABSTRACT
Photodynamic therapy (PDT) leads to the generation of cytotoxic oxygen species that appears to stimulate several different signaling pathways, some of which lead to cell death, whereas others mediate cell survival. In this context, we observed that PDT mediated by methyl-5-aminolevulinic acid as the photosensitizer resulted in over-expression of survivin, a member of the inhibitor of apoptosis (IAP) family that correlates inversely with patient prognosis. The role of survivin in resistance to anti-cancer therapies has become an area of intensive investigation. In this study, we demonstrate a specific role for survivin in modulating PDT-mediated apoptotic response. In our experimental system, we use a DNA vector-based siRNA, which targets exon-1 of the human survivin mRNA (pSil_1) to silence survivin expression. Metastatic T47D cells treated with both pSil_1 and PDT exhibited increased apoptotic indexes and cytotoxicity when compared to single-agent treated cells. The treatment resulted in increased PARP and caspase-3 cleavage, a decrease in the Bcl-2/Bak ratio and no participation of heat shock proteins. In contrast, the overexpression of survivin by a survivin-expressed vector increased cell viability and reduced cell death in breast cancer cells treated with PDT. Therefore, our data suggest that combining PDT with a survivin inhibitor may attribute to a more favorable clinical outcome than the use of single-modality PDT.
Subject(s)
Breast Neoplasms/therapy , Inhibitor of Apoptosis Proteins/antagonists & inhibitors , Photochemotherapy , Aminolevulinic Acid/analogs & derivatives , Aminolevulinic Acid/therapeutic use , Aminolevulinic Acid/toxicity , Apoptosis , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Caspase 3/metabolism , Cell Line, Tumor , Combined Modality Therapy , Female , Humans , Inhibitor of Apoptosis Proteins/genetics , Inhibitor of Apoptosis Proteins/metabolism , Neoplasm Metastasis , Photosensitizing Agents/therapeutic use , Photosensitizing Agents/toxicity , Poly(ADP-ribose) Polymerases/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Reactive Oxygen Species/metabolism , Survivin , bcl-2 Homologous Antagonist-Killer Protein/metabolismABSTRACT
A hybrid PDMS/glass microfluidic system for evaluation of the efficiency of photodynamic therapy is presented. 5-aminolevulinic acid (ALA) was used as a precursor of photosensitizer. The geometry of the microdevice presented in this paper enables to test different concentrations of the photosensitizer in a single assay. The viability of the A549 cells was determined 24 h after PDT procedure (irradiation with light which induced a photosensitizer accumulated in carcinoma cells, λ=625 nm). The presented results confirmed the possibility to perform the photodynamic therapy process in vitro in microscale and the possibility to assess its effectiveness. Moreover, because two identical microstructures on a single chip were performed, the microchip can be used for examination simultaneously various cell lines (carcinoma and normal) or various photosensitizers.
Subject(s)
Microfluidic Analytical Techniques/methods , Photochemotherapy , Aminolevulinic Acid/chemistry , Aminolevulinic Acid/therapeutic use , Aminolevulinic Acid/toxicity , Cell Line, Tumor , Dimethylpolysiloxanes/chemistry , Humans , Lab-On-A-Chip Devices , Lung Neoplasms/drug therapy , Microfluidic Analytical Techniques/instrumentation , Photosensitizing Agents/chemistry , Photosensitizing Agents/therapeutic use , Photosensitizing Agents/toxicity , Protoporphyrins/chemistryABSTRACT
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.
Subject(s)
Photosensitizing Agents/toxicity , Aminolevulinic Acid/toxicity , Anthracenes , Antineoplastic Agents/toxicity , Apoptosis , Cell Line, Tumor , Dihematoporphyrin Ether/toxicity , Humans , Indoles/toxicity , Mesoporphyrins/toxicity , Organometallic Compounds/toxicity , Perylene/analogs & derivatives , Perylene/toxicity , PhotochemotherapyABSTRACT
Photodynamic therapy (PDT) is a promising antitumor treatment strategy. However, effectiveness of PDT is limited due to an initiation of rescue responses in tumor cells, including the induction of heme oxygenase-1 (HO-1). Furthermore, the main sources of free radical production in PDT-induced oxidative stress are not clear. Here, human melanoma cells were loaded with the photosensitizer 5-aminolevulinic acid and exposed to non-thermal light of 420-800 nm at different doses. It was shown that inhibition of HO-1 activity by zinc protoporphyrin IX increased PDT-induced cytotoxicity in a dose-dependent manner. Interestingly, the cytotoxic effects were not diminished by the simultaneous application of the iron chelator desferrioxamine. Importantly, PDT together with non-toxic doses of hemin increased the number of dead cells. From these results can be concluded that heme but not iron act as the main source of free radicals in PDT treatment. This is supported by the fact that during PDT ferritin is readily up-regulated, able to bind excess iron formed by the HO-1 action. The combined treatment of photosensitizers with HO-1 inhibitors might increase the effectiveness of PDT in tumor treatment.
Subject(s)
Aminolevulinic Acid/toxicity , Heme/metabolism , Light , Photosensitizing Agents/toxicity , Aminolevulinic Acid/chemistry , Cell Line, Tumor , Deferoxamine/pharmacology , Heme Oxygenase-1/metabolism , Humans , Iron/metabolism , Iron Chelating Agents/pharmacology , Photochemotherapy , Photosensitizing Agents/chemistry , Protoporphyrins/metabolism , Reactive Oxygen Species/metabolismABSTRACT
Pept2 knockout mice are an important tool to evaluate the evolving role and relevance of this proton-coupled oligopeptide transporter beyond drug disposition, where the transporter also modulates the pharmacodynamic and toxicodynamic effects of drug substrates. Our in vivo studies with glycylsarcosine in Pept2 knockout mice have established "proof of concept" that PEPT2 can have a significant effect on dipeptide disposition. Subsequent studies with the aminocephalosporin antibiotic cefadroxil have shown relevance to pharmacology and infectious disease. Finally, studies with the endogenous peptidomimetic 5-aminolevulinic acid have demonstrated relevance to toxicology in the framework of porphyria- and lead-induced neurotoxicity. These studies have consistently demonstrated the dual action of PEPT2 with respect to its apical localization in choroid plexus epithelium and kidney in: 1) effluxing substrates from CSF into choroid plexus, thereby affecting regional pharmacokinetics in brain; and 2) reabsorbing substrates from renal tubular fluid into proximal tubules, thereby affecting systemic pharmacokinetics and exposure. Moreover, these studies have shown that the regional effect of PEPT2 in limiting substrate concentrations in the CSF is more dramatic than its effect in increasing systemic exposure. In the case of 5-aminolevulinic acid, such regional modulation of drug disposition translates directly into significant changes in neurotoxicity.