Aminolevulinic Acid Concentration Research Articles

Synthetic and Natural Vitamin C Modulation of Leaded Paint-Induced Nephrotoxicity of Automobile Painters in Ile-Ife, Nigeria.

Introduction: Lead (Pb) occupational exposure in painters has been documented to be contacted via additives in paints due to its many important properties and vitamin C has been the most widely studied when it comes to Pb- induced oxidative stress.  Aim: This study aimed at the use of freshly squeezed orange-juice due to its accessibility in investigating the modulating role of synthetic and natural vitamin-C on leaded paint-induced nephrotoxicity of automobile painters.  Study Design and Methods: Sixty (60) male automobile painters were consecutively selected and divided equally into 2 groups. Vitamin-C and orange juice were administered daily to painters for 4 weeks at dosage levels of 200 and 184 mg/day respectively. Thirty (30) male non-painters constituted the control group. Orange juice vitamin-C content was assessed by titrimetric method and synthetic vitamin-C served as the standard drug. Renal biomarkers and reduced glutathione (GSH) were done by Colorimetry. Urine aminolevulinic acid (ALA) and Pb were assessed by ELISA technique and atomic absorption spectrophotometry respectively. Phytochemical screenings (quantitative/qualitative) and proximate analysis were done using standard methods. Data were analyzed using Pearson’s correlation coefficient and One-way Analysis of Variance (ANOVA) followed by Tukey’s post-hoc test for pairwise comparison. Statistical significance was p< 0.05.  Results: Baseline results at 0-week of orange juice administered group showed a significantly (P<0.05) higher serum Pb, urea and creatinine compared to non- painters. Also, their urine baseline results at 0-week showed a significantly (P<0.05) higher levels of ALA and GSH compared to non-painters. Orange juice administration at 4 weeks showed significant (P<0.05) reductions in concentrations of lead, urea, and creatinine in serum, decreased concentrations of GSH and ALA in urine but increased urine Pb compared to baseline. However, compared with baseline, after 4 weeks of vitamin-C supplementation, serum Pb, urine GSH, and urine ALA were significantly (P<0.05) reduced and urine Pb significantly (P<0.05) increased. A positive correlation was observed at 2-weeks of taking orange- juice between serum lead and urine ALA (r= 0.703) and GSH (r= 0.913) but 4-week positive correlation between urea and urine GSH (r= 1.000). A negative correlation was observed at 2-week of taking vitamin-C between serum creatinine and urine lead (r= -0.857) while 4-week a negative correlation was observed between urine GSH and urine lead (r= -0.743). Presence of tannin, phenol, saponin, alkaloid, and flavonoid was detected in orange juice.  Conclusion: Orange juice administration conferred significant amelioration to renal and lead toxicity biomarkers by 4 weeks. The presence of phytochemicals suggests why orange juice may be a viable alternative in amelioration of toxic effects of leaded paint among automobile painters.

Modifications of DAMPs levels in extracellular environment induced by aminolevulinic acid-based photodynamic therapy of esophageal cancer cells

Purpose Immunogenic cell death plays an important role in anticancer treatment because it combines cell death with appearance of damage associated molecular patterns that have the potential to activate anticancer immunity. Effects of damage associated molecular patterns induced by aminolevulinic acid-based photodynamic therapy were studied mainly on dendritic cells. They have not been deeply studied on macrophages that constitute the essential component of the tumor microenvironment. The aim of this study was to analyze features of esophageal cancer cell death in relation to release capacity of damage associated molecular pattern species, and to test the effect of related extracellular environmental alterations on macrophages. Material and methods Esophageal Kyse 450 carcinoma cells were subjected to aminolevulinic acid-based photodynamic therapy at different concentrations of aminolevulinic acid. Resting, IFN/LPS and IL-4 macrophage subtypes were prepared from monocytic THP-1 cell line. Cell death features and macrophage modifications were analyzed by fluorescence-based live cell imaging. ATP and HMGB1 levels in cell culture media were determined by ELISA assays. The presence of lipid peroxidation products in culture media was assessed by spectrophotometric detection of thiobarbituric acid reactive substances. Results Aminolevulinic acid-based photodynamic therapy induced various death pathways in Kyse 450 cells that included features of apoptosis, necrosis and ferroptosis. ATP amounts in extracellular environment of treated Kyse 450 cells increased with increasing aminolevulinic acid concentration. Levels of HMGB1, detectable by ELISA assay in culture media, were decreased after the treatment. Aminolevulinic acid-based photodynamic therapy induced lipid peroxidation of cellular structures and increased levels of extracellular lipid peroxidation products. Incubation of resting and IL-4 macrophages in conditioned medium from Kyse 450 cells treated by aminolevulinic acid-based photodynamic therapy induced morphological changes in macrophages, however, comparable alterations were induced also by conditioned medium from untreated cancer cells. Conclusion Aminolevulinic acid-based photodynamic therapy leads to alterations in local extracellular levels of damage associated molecular patterns, however, comprehensive studies are needed to find whether they can be responsible for macrophage phenotype modifications.

Effects of Photodynamic Therapy Using 5 -Aminolevulinic Acid (ALA) Loaded Acrylic Nanoparticles (ANPs) on HaCaT Cells.

ALA-PDT (5-aminolevulinic acid photodynamic therapy) is a central modality in the treatment of skin diseases. Increasing the bioavailability of ALA remains a critical issue. With this in mind, our study explores a novel route of ALA delivery by loading acrylic nanoparticles (ANPs). ALA-ANPs were synthesized by emulsion polymerisation and characterised by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). The effects of ALA-ANPs on HaCaT cell line were evaluated, including characteristics, morphological changes, protoporphyrin IX (PpIX) fluorescence kinetics, reactive oxygen species (ROS) levels, mitochondrial membrane potential and ki67 expression in these cells. The ANPs had uniform sizes, smooth surfaces and excellent light transmittance, with diameters of 150-200 nm. In contrast, the ALA - ANPs had uneven surfaces and poor light transmittance, with diameters of 220-250 nm. During 12 hours of co-incubation of HaCaT cells with ALA, the intracellular accumulation of PpIX increased over time. Notably, after 6 hours of incubation, PpIX levels induced by 1.81 mg/mL ALA-ANPs exceeded those induced by 1.0 mM ALA (p < 0.01). CCK-8 results showed a positive correlation between PDT-induced inhibition of HaCaT cell proliferation and ALA concentration when ALA concentration remained below 2.0 mM. Compared to the 1.0 mM ALA group, the 1.81 mg/mL ALA-ANPs group showed decreased mitochondrial membrane potential, ki67 immunofluorescence intensity and cell proliferation. In contrast, ROS levels were significantly increased in the 1.81 mg/mL ALA-ANPs group (p < 0.01). Loading ANPs provide improved stability and potency for ALA. The ALA-ANPs-PDT approach has superior inhibitory effects on HaCaT proliferation in vitro.

Suppression of resistance to aminolevulinic acid-based photodynamic therapy in esophageal cell lines by administration of iron chelators in collagen type I matrices

Purpose Photodynamic therapy (PDT) utilizes visible light to activate the cytotoxic effects of photosensitizing drugs. PDT protocols require optimization to overcome treatment resistance and induce a beneficial anti-tumor immune response. The aim of this study was to examine the possibility to suppress the resistance of esophageal cell lines to aminolevulinic acid (ALA)-PDT by administration of iron chelators to induce sufficient cell cytotoxicity under pathophysiologically relevant conditions, mimicking the advanced stages of cancer. Materials and methods Effects of ALA-PDT in combination with iron chelators were compared in three esophageal cell lines in conventional monolayers and in 3 D cultures based on collagen type I. Modified colony assay and fluorescence-based live cell imaging, respectively were applied. The latter was used also to test the capability of pre-polarized macrophages to interact with cancer cells subjected to ALA-PDT with or without iron chelators. Results Iron chelators were effective in the enhancement of ALA-PDT in all cell lines under both culture conditions. Fluorescence evaluation of cell viability in 3 D cultures indicated the contribution of apoptotic cell death after ALA-PDT, both with and without iron chelators. Engulfment of remnants of dead cancer cells by macrophages in 2 D cultures was indicated, however, the interaction between macrophages and cancer cells in 3 D cultures subjected to ALA-PDT with or without iron chelators was not present. Conclusions The potential of iron chelators to enhance ALA-PDT was maintained in 3 D collagen matrices. Although PDT dose (ALA concentration, light exposure time) required modification in a cell line-dependent manner to achieve a comparable effect of PDT alone in conventional monolayers and in collagen matrices, the potential of iron chelators to suppress the resistance of esophageal cells to ALA-PDT was not influenced by a fibrillar collagen matrix.

Interstitial photodynamic therapy: Light fractionation effects on a preclinical model of glioblastoma

s / Photodiagnosis and Photodynamic Therapy 12 (2015) 325–375 347 natural (sunlight) light sources. Fluorescence imageswere taken to locate the distribution of the curcumin in themosquito larvae. Both curcumins derivative exhibited photoactivity, reaching 100% larval mortality after exposure to different light sources shown above. http://dx.doi.org/10.1016/j.pdpdt.2015.07.087 Study of ALA andMALmixtures in pig skin: PpIX production Alessandra Keiko Lima Fujita, Priscila Fernanda Campos de Menezes, Phamilla Gracielli Sousa Rodrigues, Michelle Barreto Requena, Cristina Kurachi, Andre Escobar, Rozana Wendler da Rocha, Andrigo Barboza de Nardi, Vanderlei S. Bagnato Sao Carlos Institute of Physics, University of Sao Paulo, Brazil Topical PDT using ALA (aminolevulinic acid) or methyl-ALA (MAL) as precursor of protoporphyrin IX (PpIX) shows some limitations. ALA is more hydrophilic than MAL, resulting in differences at the molecule interaction with the cells. The goal of study is evaluated the optimization of topical PDTusingALA andMAL in differentmixtures. This studywas done in vivo in porcine skinmodels using 7 categories of ALA andMALmixtures. The cream in different mixtures was applied and an occlusive dressing was placed. PpIX production in a porcine skin model was monitored using widefield fluorescence imaging and fluorescence spectroscopy collected at skin surface until 5 h of treatment. Seven types of mixture were tested, from 0 to 100% combinations, varying on 20%. The imaging analyses, showed the best results in PpIX productionwithmixtures from 40% to 80% of ALA. Both fluorescence techniques showed that the higher relative production of PpIX was present when the ALA concentration was of 40–80%. The investigation of the PpIX production as a function of the tissue depth is being performed. These results can be useful in Clinical Applications of PDT using topical ALA and M-ALA (Financial support: CNPq). http://dx.doi.org/10.1016/j.pdpdt.2015.07.088 Interstitial photodynamic therapy: Light fractionation effects on a preclinical model of glioblastoma Henri-Arthur Leroy, Maximilien Vermandel, Bertrand Leroux, Serge Mordon, Nicolas Reyns INSERM U1189 – ONCO-THAI Lille, Department of Neurosurgery/University Hospital, University of Lille,

Study of ALA and MAL mixtures in pig skin: PpIX production

s / Photodiagnosis and Photodynamic Therapy 12 (2015) 325–375 347 natural (sunlight) light sources. Fluorescence imageswere taken to locate the distribution of the curcumin in themosquito larvae. Both curcumins derivative exhibited photoactivity, reaching 100% larval mortality after exposure to different light sources shown above. http://dx.doi.org/10.1016/j.pdpdt.2015.07.087 Study of ALA andMALmixtures in pig skin: PpIX production Alessandra Keiko Lima Fujita, Priscila Fernanda Campos de Menezes, Phamilla Gracielli Sousa Rodrigues, Michelle Barreto Requena, Cristina Kurachi, Andre Escobar, Rozana Wendler da Rocha, Andrigo Barboza de Nardi, Vanderlei S. Bagnato Sao Carlos Institute of Physics, University of Sao Paulo, Brazil Topical PDT using ALA (aminolevulinic acid) or methyl-ALA (MAL) as precursor of protoporphyrin IX (PpIX) shows some limitations. ALA is more hydrophilic than MAL, resulting in differences at the molecule interaction with the cells. The goal of study is evaluated the optimization of topical PDTusingALA andMAL in differentmixtures. This studywas done in vivo in porcine skinmodels using 7 categories of ALA andMALmixtures. The cream in different mixtures was applied and an occlusive dressing was placed. PpIX production in a porcine skin model was monitored using widefield fluorescence imaging and fluorescence spectroscopy collected at skin surface until 5 h of treatment. Seven types of mixture were tested, from 0 to 100% combinations, varying on 20%. The imaging analyses, showed the best results in PpIX productionwithmixtures from 40% to 80% of ALA. Both fluorescence techniques showed that the higher relative production of PpIX was present when the ALA concentration was of 40–80%. The investigation of the PpIX production as a function of the tissue depth is being performed. These results can be useful in Clinical Applications of PDT using topical ALA and M-ALA (Financial support: CNPq). http://dx.doi.org/10.1016/j.pdpdt.2015.07.088 Interstitial photodynamic therapy: Light fractionation effects on a preclinical model of glioblastoma Henri-Arthur Leroy, Maximilien Vermandel, Bertrand Leroux, Serge Mordon, Nicolas Reyns INSERM U1189 – ONCO-THAI Lille, Department of Neurosurgery/University Hospital, University of Lille,

The effectivity of ALA-PDT using IPL evaluated by image and histological analysis using porcine skin models in vivo

s / Photodiagnosis and Photodynamic Therapy 12 (2015) 325–375 347 natural (sunlight) light sources. Fluorescence imageswere taken to locate the distribution of the curcumin in themosquito larvae. Both curcumins derivative exhibited photoactivity, reaching 100% larval mortality after exposure to different light sources shown above. http://dx.doi.org/10.1016/j.pdpdt.2015.07.087 Study of ALA andMALmixtures in pig skin: PpIX production Alessandra Keiko Lima Fujita, Priscila Fernanda Campos de Menezes, Phamilla Gracielli Sousa Rodrigues, Michelle Barreto Requena, Cristina Kurachi, Andre Escobar, Rozana Wendler da Rocha, Andrigo Barboza de Nardi, Vanderlei S. Bagnato Sao Carlos Institute of Physics, University of Sao Paulo, Brazil Topical PDT using ALA (aminolevulinic acid) or methyl-ALA (MAL) as precursor of protoporphyrin IX (PpIX) shows some limitations. ALA is more hydrophilic than MAL, resulting in differences at the molecule interaction with the cells. The goal of study is evaluated the optimization of topical PDTusingALA andMAL in differentmixtures. This studywas done in vivo in porcine skinmodels using 7 categories of ALA andMALmixtures. The cream in different mixtures was applied and an occlusive dressing was placed. PpIX production in a porcine skin model was monitored using widefield fluorescence imaging and fluorescence spectroscopy collected at skin surface until 5 h of treatment. Seven types of mixture were tested, from 0 to 100% combinations, varying on 20%. The imaging analyses, showed the best results in PpIX productionwithmixtures from 40% to 80% of ALA. Both fluorescence techniques showed that the higher relative production of PpIX was present when the ALA concentration was of 40–80%. The investigation of the PpIX production as a function of the tissue depth is being performed. These results can be useful in Clinical Applications of PDT using topical ALA and M-ALA (Financial support: CNPq). http://dx.doi.org/10.1016/j.pdpdt.2015.07.088 Interstitial photodynamic therapy: Light fractionation effects on a preclinical model of glioblastoma Henri-Arthur Leroy, Maximilien Vermandel, Bertrand Leroux, Serge Mordon, Nicolas Reyns INSERM U1189 – ONCO-THAI Lille, Department of Neurosurgery/University Hospital, University of Lille,

Evidence-based review of photodynamic therapy in the treatment of acne.

Photodynamic therapy (PDT) has been used for acne, however, the efficacy and safety need to be determined. To assess the effects and safety of PDT for acne using an evidence-based approach. Randomized controlled trials (RCTs) on the treatment of acne with PDT were identified by searching PubMed, CNKI and the Cochrane Library. A total of 14 RCTs involving 492 patients were included. Photosensitizers included aminolevulinic acid (ALA), methylaminolevulinate (MAL), and indole-3-acetic acid (IAA). Light sources included red light, pulsed dye laser (PDL), intense pulsed light (IPL), long-pulsed dye laser (LPDL) and green light. The PDT protocols, including ALA + red light, ALA + PDL, ALA + IPL, MAL + red light, and MAL + LPDL, all showed great efficacy on inflammatory lesions. ALA + red light also had effects on non-inflammatory lesions and sebum secretion. ALA + IPL and IAA + green light significantly decreased sebum secretion. Triple treatment protocols showed great improvement on inflammatory and non-inflammatory lesions. Increasing ALA concentration, ALA incubation time, PDT sessions, dose of light source or using occlusion for photosensitizers, or a combination of other treatments with PDT may achieve greater efficacy. The common side effects of PDT were tolerable and transient. Limited evidence indicates that PDT shows good efficacy in the treatment of acne with acceptable side effects. ALA + red light was shown to be the optimal choice. However, more RCTs are needed to determine the types and concentrations of photosensitizers and light sources, and the duration of light activation and incubation.

Aminolevulinic acid derivatives-based photodynamic therapy in human intra- and extrahepatic cholangiocarcinoma cells

Hexyl-aminolevulinic acid (HALA) was compared with aminolevulinic acid (ALA) in terms of improving ALA-based photodynamic therapy (PDT) for human intra- and extrahepatic cholangiocarcinoma (CCA) HuCC-T1 and SNU1196 cells. Because of the different uptake mechanisms of HALA, a relatively higher amount of protoporphyrin IX (PpIX) was induced in the both CCA cell types at low concentrations of HALA. Furthermore, higher expression of porphobilinogen deaminase, coproporphyrinogen III oxidase, and protoporphyrinogen oxidase, the key enzymes for synthesizing PpIX in the heme biosynthetic pathway, facilitated the exuberant generation of PpIX in HuCC-T1 cells. PpIX accumulation with ALA was markedly different between the two CCA cell types. Even at lower concentrations of ALA, SNU1196 cell successfully synthesized PpIX, due to the higher expression of the ALA transporter, mammalian H +/peptide co-transporter PEPT1. Considering the difference of PEPT1 or key enzyme expression, HALA could be a very effective substitute for ALA in doing PDT for cure of CCA.

Abstract 3464: Aminolevulinic acid-mediated photodynamic therapy induces cell death in human osteosarcoma cells

Abstract Photodynamic therapy (PDT) is a localized cancer treatment that requires a photosensitizer (PS), molecular oxygen, and light to induce cell death. Aminolevulinic acid (ALA)-mediated PDT utilizes ALA as an inert precursor to the required PS. Subsequent to cellular uptake ALA is converted to a PS, protoporphyrin IX (PpIX), in the mitochondria via the heme pathway. The excitation of PpIX by light can then result in the generation of reactive oxygen species, leading to direct tumor cell death, damage to the microvasculature, and induction of local inflammatory response. Interestingly, PDT can evoke apoptotic and/or necrotic cell death pathways within the same cell population. The balance between apoptosis and necrosis depends on several parameters, including the total PDT dose, the intracellular localization of the PS, the fluence rate, and the cell type. Recent studies show that the cell death mechanism induced has a direct influence on the systemic immune response. Therefore, a better understanding of the interplay between different cell death mechanisms in response to PDT could lead to improved tumor response and could inform better therapeutic strategies. We first determined the cytotoxicity of ALA to the human osteosarcoma cell line MG-63. PDT is a relatively unexplored method of treatment for human osteosarcoma, and could represent a superior method to existing therapies. Data from our lab show that ALA is not significantly cytotoxic to MG-63 cells, and that it is efficiently converted to PpIX within 24 hours of treatment. We also demonstrate that ALA-mediated PDT significantly induces cell death in MG-63 cells, and that cell death increases with increased ALA concentration and light fluence. Our findings indicate that this cell death results from a combination of both apoptosis and necrosis, with necrosis representing the major pathway. Finally, we show that increased light fluence decreases the amount of observed apoptosis subsequent to ALA-mediated PDT. These results taken together suggest that ALA-mediated PDT can induce cell death in human osteosarcoma cells, and that most cells are dying by necrosis. Furthermore, our data suggests that by adjusting specific therapeutic parameters, we can shift the balance between separate death pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3464. doi:1538-7445.AM2012-3464

Intracutaneous ALA photodynamic therapy: Dose-dependent targeting of skin structures

Photodynamic therapy (PDT) using topical aminolevulinic acid (ALA) depends on local drug uptake, metabolism to porphyrins, and depth of light penetration using different wavelengths. Topical ALA-PDT has limited depth of drug penetration. We studied induced porphyrin distribution and PDT after intradermal ALA administration using different drug concentrations followed by high-fluence red light irradiation. Intradermal injections (∼2 mm deep) of ALA concentrations from 0.0005% to 1% were studied in swine to evaluated porphyrin fluorescence before PDT and clinical and histological damage 24 hours after PDT. Porphyrin accumulation was measured by fluorescence microscopy of frozen section. PDT was performed 3 hours after intradermal injections using a 635 nm LED array at a fluence of 200 J/cm2 . Skin responses to PDT were observed grossly and by histology (blind evaluation). Intradermal ALA caused porphyrin accumulation in epidermis, hair follicles (HF), sebaceous glands (SG), sweat glands (eccrine glands, EG and apocrine glands, AG), and subcutaneous fat. Significant differences of fluorescence intensity were observed between different skin structures (P < 0.05), but there was no significant difference comparing HF to SG; epidermis with either HF or SG; and dermis with fat (P > 0.05). Intradermal ALA is potent. ALA concentrations ≥0.25% followed by red light exposures caused a very intense vascular PDT reaction. Moderate doses of injected ALA concentration (∼0.06%), selectively targeted EG. Low doses (≤0.016%) targeted fat; producing fat necrosis with minimal inflammation, manifested both clinically and histologically. In contrast to topical ALA-PDT, intradermal ALA-PDT can effectively photosensitize deep skin structures. Potentially, intradermal ALA-PDT using various ALA concentrations may be useful for treating vascular lesions (malformations, hemangiomas, tumors), EG/AG disorders, fat or deep targets in skin.

Inhibition of δ-aminolevulinic acid dehydratase activity by cadmium in excised etiolated maize leaf segments during greening

Supply of 0.1&amp;ndash;0.5 mmol CdCl&lt;sub&gt;2&lt;/sub&gt; inhibited &amp;delta;-aminolevulinic acid dehydratase (EC 4.2.1.24, ALAD) activity and total chlorophylls in excised etiolated segments of maize leaves during greening. Due to cadmium supply &amp;delta;-aminolevulinic acid (ALA) content was reduced significantly at 0.5 mmol Cd only. Also the Cd treatment decreased the protein content and accumulated significantly the Cd in the tissue. Significant correlation between Cd accumulation in the leaves and various parameters measured is observed, with the R-squared values being 0.727 with ALAD activity, 0.885 with ALA content, 0.902 with total chlorophylls and 1.00 with proteins. The % inhibition of ALAD activity by Cd was decreased in the presence of nitrogenous compounds, glutamine and NH&lt;sub&gt;4&lt;/sub&gt;NO&lt;sub&gt;3&lt;/sub&gt; and the observed inhibition was 25% and 16%, respectively. More substantial reduction in % inhibition of enzyme activity by Cd was observed during treatment with glutathione, a ubiquitous thiol and levulinic acid, a competitive inhibitor of ALAD, with the inhibition being only 2% and 4%, respectively. Supply of some essential metal ions, such as Mg, Zn, and Mn, also reduced the % inhibition of enzyme activity by Cd. Inclusion of varying concentrations of ALA during assay also affected the % inhibition of enzyme activity by Cd showing an increased inhibition from 17% to 53% with increasing ALA concentration. It is suggested that Cd inhibits ALAD activity by affecting the ALA binding to the enzyme and/or disrupting thiol interaction.

The role of sensitivity of ALA (PpIX)-based PDT on Human embryonic kidney cell line (HEK293T)

Present study evaluates the effects of photodynamic therapy (PDT) with aminolevulinic acid (5-ALA) as photo sensitizer using Human embryonic kidney (HEK293T) cell line as an experimental model. Porphyrins derivatives are used as active cytotoxic antitumor agents in PDT. Above mentioned cell line were irradiated with red light (a diode laser, λ = 635 nm) at different doses (0–160 J/cm2) of light. The influence/effectiveness of incubation time, various concentrations of aminolevulinic acid (5-ALA) and light doses on the cellular viability was studied. HEK293T cells were deliberated by exposing the ALA-PpIX (0–1000 μg/ml) of concentrations. The optimal uptakes of photosensitizer (PS) in cell lines were investigated by means of spectro photo metric measurements. Cells viability was determined by means of neutral red assay (NRA). It was observed that alone, neither photosensitizer nor light dose have significant effect on cells viability, but optimal concentration of PS along with suitable dose of light exhibit effective impact on the viability of cell. Our results showed that light doses of 40 J/cm2 demonstrates effective PDT outcome for HEK293T cell line when incubated with 400 μg/ml, with wrapping up view that HEK293T cell line is very sensitive to ALA-mediated PDT as compared to cell line published in our data. At the end results has been verified by using reactive oxygen species (ROS) measure test.

Photodynamic therapy with different concentrations of aminolevulinic acid in the treatment of moderate to severe acne.

Objective To investigate the efficacy and safety of photodynamic therapy using different concentrations of aminolevulinic acid(ALA)in the treatment of moderate to severe acne.Methods One hundred and eighty patients with moderate to severe acne were randomly divided into 4 groups to apply different concentrations(5%, 10%, 15%, 20%)of ALA to acne lesions on the right side and placebo agent on the left side of faces as control.The whole face was irradiated with red light once a week for 4 sessions.The subsidence of lesions was evaluated by a dermatologist at the baseline and on week 2, 1 month, 3 month, and 6 month after the last treatment.Adverse effects were recorded at each follow-up visit.Results At the end of treatment, a stronger improvement was observed in ALA-treated lesions compared with those treated with red light alone(P ＜ 0.05).Six months after the last treatment, the effective rates were 79.55%, 88.37%, 95.24%,97.73% in lesions treated with ALA of 5%, 10%, 15% and 20% , respectively, significantly higher than those treated with placebo(11.51%, 9.82%, 13.50%, 10.07%, all P＜ 0.05).With the elevation of ALA concentration, the effective rate together with incidence of adverse reactions including erythematous swelling and hyperpigmentation increased(all P ＜ 0.05).No scarring was noted.Conclusions The clinical effect of ALA-PDT is superior to that of red light alone in the treatment of moderate to severe ache.In view of effectiveness and safety, ALA-PDT using 10% or 15% ALA is recommended for the treatment of moderate to severe acne. Key words: Acne vulgaris; Aminolevulinic acid; Photochemotherapy; Treatment outcome

Characterisation of liposomes containing aminolevulinic acid and derived esters

Liposomes of different compositions have been designed to improve delivery of aminolevulinic acid (ALA) and its esterified derivatives ALA-Hexyl ester (He-ALA) and ALA-Undecanoyl ester (Und-ALA) for its use in photodynamic therapy (PDT). Egg yolk phosphatidyl choline (PC), phosphatidic acid (PA) and phosphatidyl glycerol (PG) were employed in the preparation of the liposomes. Sonicated vesicles composed of PC, PC–PG (80:20) or PC–PA (80:20) containing ALA or derivatives were obtained and purified by a minicolumn centrifugation method. PC liposomes presented encapsulation percentages around 6% for 2 mM ALA, 13% for 2 mM He-ALA and 51% for 2 mM Und-ALA. The addition of PG or PA to the formulation, resulted in an increased entrapment: 19% for 2 mM ALA, 69% for 2 mM He-ALA and 87% for 2 mM Und-ALA in PC–PG liposomes and 21% for 2 mM ALA, 60% for 2 mM He-ALA and 87% for 2 mM Und-ALA in PC–PA liposomes. Higher concentrations of ALA or derivatives resulted in lower percentages of entrapment. The three formulations containing ALA or derivatives were stable up to 1 week upon storage at 4 °C. However, upon dilution with medium, ALA leaked from the liposomes, while on the contrary, He-ALA was highly retained, being therefore a good choice for its use in PDT. The stability of Und-ALA upon dilution could not be tested, but Und-ALA proved to have the highest entrapment efficacy.

Phototoxicity of 5-Aminolevulinic Acid in the HeLa Cell Line as an Indicative Measure of Photodynamic Effect After Topical Administration to Gynecological Lesions of Intraepithelial Form

The depth-resolved pattern of aminolevulinic acid (ALA) concentration in excised vaginal tissue was determined after in vitro application of an ALA-loaded bioadhesive patch. From this data, the tissue concentration of ALA achievable at a specified depth from the surface could be related to the concentration needed to elicit a photodynamic effect in a model gynecological tumor cell line (HeLa). Excised vaginal tissue was mounted in a modified Franz diffusion cell and exposed to a water-soluble, ALA-loaded, bioadhesive patch. After a period of time, the tissue was cryostatically sectioned and the stratal concentration of radiolabeled ALA determined using scintillation spectroscopy. HeLa cells were cultured in media containing specific concentrations of ALA and exposed to standard photodynamic protocols of light exposure. An ALA concentration of 65.6 mM was achievable at 2.375 mm from the tissue surface after application of ALA-loaded patch. The photodynamic effectiveness of this concentration was demonstrated in HeLa with exposure to concentrations exceeding 1.0 mM ALA bringing about reductions in viable cell numbers by 90%. An enhancement of PpIX production using adjunctive EDTA over the clinically relevant 4 h application time interval was shown to be minimal in HeLa. Instead, PpIX production was more closely correlated with ALA concentration, with 100 mM ALA producing approximately 3100 ng PpIX mg(-1) protein in the same time period. Given that vaginal intraepithelial neoplasias can extend to 2.0 mm from the lesion surface, the ALA permeability derived from a bioadhesive patch is sufficient to induce photosensitization suitable for light induced destruction at deep sites of this type of lesion.

Evaluation of the penetration of 5-aminolevulinic acid through basal cell carcinoma: a pilot study.

Aminolevulinic acid (ALA) is a charged, hydrophilic molecule that penetrates poorly through cellular structures. This property has been implicated in the poor clinical response of non-superficial basal cell carcinomas (BCCs) to photodynamic therapy (PDT). Release of ALA hydrochloride from a 20% w/w formulation was found to be incomplete and that approximately 36.8% of the total dose is released during the application period of 4 h. Using scintillation spectroscopy and a precise tissue sectioning protocol, it was demonstrated that depths of penetration of at least 2 mm from the lesion surface had been reached. Using cumulative stratal ALA concentrations, it was found that 10% of the total applied dose permeated into the lesion. In spite of this, comparisons drawn with photodynamic concentrations used in tissue culture work reported elsewhere revealed that estimations of the ALA concentration at 2 mm were sufficient to elicit a possible therapeutic response. Results from this work question the reasons given for poor outcomes of PDT in nodular BCC based solely on depth as a hindering factor.

Photodynamic Therapy induced Cell Death using ALA and 632nm Diode Laser in A549 Lung Cancer Cells

Background : Photodynamic therapy (PDT) is a new therapeutic method aimed at the selective destruction of cancer cells. The outcome is death of cancer cells through apoptosis or necrosis. The aim of this study was to investigate the characterization of PDT induced cell death in A549 lung cancer cells. Materials and methods : A549 cells were used as the lung cancer cell. 5 aminolevulinic acid (ALA) was used as the photosensitizer and a 632nm diode laser (Biolitec, Germany) as the light source. Cells were incubated with various concentrations of ALA. The 632nm diode laser was then administered for various laser irradiation times. The treated cells were incubated with 24, 48 and 72 hours. The cell viabilities were measured using the crystal violet assay and light microscopy. To observe the cell death mechanism after PDT, cells were observed under fluorescence microscopy after double staining with Hoechst 33342 and propium iodide after PDT. Results : In the crystal violet assay at 24 hours after PDT with a laser irradiation power, the cell viabilities were , , and with ALA concentrations of 10, 100, and , respectively. In crystal violet assay at 24 hours after PDT with of ALA, the cell viabilities were , , and with 1.6, 3.2 and laser irradiation powers, respectively. However, increasing the interval time after PDT did not change the cell viabilities. In the apoptosis assay, photodynamic therapy was inducing the apoptotic cell death. Conclusions : This study shows the apoptotic anticancer effect of photodynamic therapy in A549 lung cancer cells. However, further evaluations with other cancer cells and photosensitizers are necessary.

PpIX Fluorescence Kinetics and Increased Skin Damage after Intracutaneous Injection of 5-Aminolevulinic Acid and Repeated Illumination

Photodynamic therapy with topically applied 5-aminolevulinic acid is used successfully for superficial skin lesions. The results for thicker, nodular lesions are less favorable. The method of aminolevulinic acid administration, the concentrations of aminolevulinic acid, and the irradiation schemes used so far have not been investigated thoroughly. As aminolevulinic acid photodynamic therapy has high potential for the increasing problem of skin cancer, we investigated both visually and histopathologically the photodynamic-therapy-induced skin damage after intracutaneous administration of aminolevulinic acid in normal porcine skin. We also investigated the kinetics of the aminolevulinic-acid-induced photosensitizer protoporphyrin IX fluorescence after irradiation in relation to fluence and irradiance. Finally we investigated the effect on photodynamic-therapy-induced damage of a fractionated irradiation. This study demonstrates that intracutaneous administration of aminolevulinic acid leads to higher fluorescence levels and thus to formation of higher protoporphyrin IX concentrations than topical application of aminolevulinic acid cream. The peak level of protoporphyrin IX after intracutaneous administration of aminolevulinic acid is reached earlier than after topical administration. The comeback of fluorescence, indicating re-synthesis of protoporphyrin IX after irradiation, is inhibited with increasing fluence. Photodynamic-therapy-induced damage increases with increasing fluence, but is independent of the irradiance. Finally, the photodynamic-therapy-induced skin damage seems to be greater after fractionated irradiations with two equal light fractions of 15 J per cm2 separated by a dark interval of 2 h.

Pharmacokinetics of aminolevulinic acid after intravesical administration to dogs.

To examine the stability and systemic absorption of aminolevulinic acid (ALA) in dogs during intravesical administration. Nine dogs received an intravesical dose of ALA either with no prior treatment, after receiving ammonium chloride for urinary acidification, or after receiving sodium bicarbonate for urinary alkalinization. Urine and blood samples collected during and after administration were monitored for ALA using an HPLC assay developed in our laboratories. Concentrations of pyrazine 2,5-dipropionic acid, the major ALA degradation product, and radiolabeled inulin, a nonabsorbable marker for urine volume, were also determined. Less than 0.6% of intravesical ALA doses was absorbed into plasma. Urine concentrations decreased to 37% of the initial concentration during the 2 hour instillation. Decreases in urinary ALA and radiolabeled inulin concentrations were significantly correlated, indicating that urine dilution accounted for over 80% of observed decreases in urinary ALA. ALA conversion to pyrazine 2,5-dipropionic acid was negligible. These studies demonstrate that ALA is stable and poorly absorbed into the systemic circulation during intravesical instillation. Future studies utilizing intravesical ALA for photodiagnosis of bladder cancer should include measures to restrict fluid intake as a means to limit dilution and maximize ALA concentrations during instillation.