In-vitro investigation of ALA-induced protopoyphyrin IX

doi:10.1016/S1011-1344(96)07358-7

Journal of Photochemistry and Photobiology B: Biology

Volume 36, Issue 2, November 1996, Pages 121-126

https://doi.org/10.1016/S1011-1344(96)07358-7 Get rights and content

Abstract

Protoporphyrin IX (PpIX) induced endogenously by δ-aminolevulinic acid (ALA), can be used to destroy photodynamically tumor cells. The influence of several parameters on the PpIX formation of human skin fibroblasts was investigated by fluorescence spectrophotometry. The PpIX formation increases (1) with the pH value of ALA, (2) with the ALA incubation time in a moderate sigmoidal manner, and (3) with the ALA concentration up to 700 μg ml⁻¹. Other parameters, such as cell washing procedures, have no influence on the PpIX production. ALA has to be applied in a concentration 30 times higher than external protoporphyrin IX and Photosan 3 in order to produce the same cytotoxic damage. Protoporphyrin bleaching and photoproduct generation at 646 nm was observed. Additional information about intracellular PpIX formation kinetics and its topographically correlation to cell structures was gained by a CCD camera mounted on a fluorescence microscope. A few minutes after the onset of incubation with ALA, PpIX generation is observed in the mitochondria, followed by relocalization in the cytoplasm and the nuclear membrane.

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Photodynamic effect of protoporphyrin IX in gliosarcoma 9l/lacZ cell line
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Citation Excerpt :
The authors also observed an intracellular aggregation from 1000 µg/mL, interfering with the biophysical characteristics of the drug, thus reducing its efficiency. Chung et al. (2012) also observed the saturation in the training of PpIX when used above 5 mM (∼800 µg/mL) [25, 26]. In a study by Liu et al. (2011), a hematoporphyrin monomethyl ether (HMME) was used in a canine breast cancer lineage (CHMm), using the trypan blue exclusion test as the cell death evaluation method.
Photodynamic Therapy (PDT) is an oncologic treatment, producing reactive oxygen species (ROS) to induce the death of cancer cells. This study aimed to evaluate the action of PDT on gliosarcoma cells, using protoporphyrin IX as PS by incubation with the precursor aminolevulinic acid (ALA). An LED device was used with a light dose of 10 J/cm². The success of the therapy proved to be dependent on the concentration of ALA, and an incubation time of 4 h required for an effective response. Cell death was prevalent due to necrosis when assessed 18 h post-PDT. ALA proved to be an option to PDT in cells of the 9 L/lacZ, with the protocol tested.
Efficiency of aminolevulinic acid (ALA)-photodynamic therapy based on ALA uptake transporters in a cell density-dependent malignancy model
2021, Journal of Photochemistry and Photobiology B: Biology
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This finding coincided with results from Fig. 3a whereby ALA-PDT was observed to be more effective at high cell density/malignancy condition. Several papers have reported that PpIX fluorescence increased as cell density increased in vitro while this effect was more profound in malignant cells compared to normal cells [24–30]. Another study by Juzeniene et al., (2009) suggest that ALA-induced PpIX accumulation was efflux out of the cell at a higher rate when cultured in low density than in high density [31,32].
The effectiveness of the conventional chemotherapy for cancer are compromised as the cancer cells advances in their malignancy level as they acquired drug resistance. In this study, we aimed to evaluate the efficiency of aminolevulinic acid-photodynamic therapy (ALA-PDT) against cancer of various malignancy levels, indicated by the expression level of receptor associated nuclear factor-κB ligand (RANKL), through the expression levels of ALA uptake transporters. We established a malignancy model by gradually increasing the cell density of cancer cells. Western blotting was used to study the expression levels of RANKL, ALA uptake transporters and the cell density-dependent Yes-associated protein (YAP) under different cell densities. The amount of protoporphyrin (PpIX) produced and cell viability were then studied using high performance liquid chromatography (HPLC) and ALA-PDT assay. Our study showed that the amount of PpIX production doubled in high cell density/cancer malignancy cultures and the effectiveness of ALA-PDT when subjected to light irradiation at 635 nm are significantly at higher cancer malignancy. We observed that the expression levels of ALA uptake transporters and YAP correlated with higher cell density/cancer malignancy, suggesting a possible relationship among these three factors. These findings suggest that ALA-PDT is more effective in cancer cells of higher malignancy due to the upregulation of transporters involved in ALA uptake.
Influence of culture conditions on porphyrin production in Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis
2017, Photodiagnosis and Photodynamic Therapy
Citation Excerpt :
They are either added in their native form directly to the site of treatment, or by induced production of endogenous porphyrins by addition of 5-aminolaevulinic acid. This compound is a porphyrin precursor that bypasses the feedback control of porphyrin synthesis in the cell, resulting in high porphyrin concentrations [41,24,37]. It has been shown that certain oral bacteria, e.g. P. gingivalis and A. actinomycetemcomitans, are affected by PT without exogenous addition of a photosensitizer [4,42,18,11].
Increasing antibiotic resistance among pathogens has raised the demands for new treatment methods such as antimicrobial photodynamic therapy (aPDT) and phototherapy (PT). Experiments for investigating the effects of these methods are often performed in vitro, but the procedures for cultivation of microbes vary between different studies. The aim of this study has been to elucidate how the profile of endogenously produced porphyrins differs by changing the variables of bacteria culturing conditions.
Two oral pathogens, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis, were selected as model organisms. The contents of porphyrins and heme in the bacteria were analysed with liquid chromatography–tandem mass spectrometry when bacteria was cultivated for different lengths of time (3–9 days), upon passaging as well as when growth medium were supplemented with or without horse blood.
Both porphyrin and heme content in A. actinomycetemcomitans are highly affected by the age of the culture, and that the porphyrin profiles changes during cultivation. When cultivated colonies of A. actinomycetemcomitans were passaged onto a new, fresh growth medium a large change in porphyrin content occurred. Additional porphyrins were detected; uroporphyrin and 7-carboxylporphyrin, and the total porphyrin content increased up to 28 times. When P. gingivalis was grown on blood containing medium higher concentrations of protoporphyrin IX (2.5 times) and heme (5.4 times) were quantified compared to bacteria grown without blood.
This study demonstrate that there is a need for more standardized culturing protocols when performing aPDT and PT experiments in vitro to avoid large variations in porphyrin profiles and concentrations, the aPDT/PT target compounds, depending on the culturing conditions.
Microneedle-mediated intradermal delivery of 5-aminolevulinic acid: Potential for enhanced topical photodynamic therapy
2008, Journal of Controlled Release
Photodynamic therapy of deep or nodular skin tumours is currently limited by the poor tissue penetration of the porphyrin precursor 5-aminolevulinic acid (ALA). In this study, silicon microneedle arrays were used, for the first time, to enhance skin penetration of ALA in vitro and in vivo. Puncturing excised murine skin with 6 × 7 arrays of microneedles 270 μm in height, with a diameter of 240 μm at the base and an interspacing of 750 μm led to a significant increase in transdermal delivery of ALA released from a bioadhesive patch containing 19 mg ALA cm^− 2. Microneedle puncture enhanced ALA delivery to the upper regions of excised porcine skin but, at mean depths of 1.875 mm, ALA concentrations were similar to control values, possibly reflecting binding of ALA by tissue components. However, and importantly, in vivo experiments using nude mice showed that microneedle puncture could reduce application time and ALA dose required to induce high levels of the photosensitiser protoporphyrin IX in skin. This clearly has implications for clinical practice, as shorter application times would mean improved patient and clinician convenience and also that more patients could be treated in the same session. As ALA is expensive and degrades rapidly via a second order reaction, reducing the required dose is also a notable advantage.
Influence of solution viscosity and injection protocol on distribution patterns of jet injectors: Application to photodynamic tumour targeting
2007, Journal of Photochemistry and Photobiology B: Biology
Photodynamic therapy of deep or nodular skin tumours is currently limited by the poor tissue penetration of the porphyrin precursor 5-aminolevulinic acid (ALA) and preformed photosensitisers. In this study, we investigated the potential of jet injection to deliver both ALA and a preformed photosensitiser (meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate, TMP) into a defined volume of skin. Initial studies using a model hydrogel showed that as standoff distance is increased, injection depth decreases. As the ejected volume is increased, injection depth increases. It was also shown, for the first time, that, as injection solution viscosity was increased, for a given injection setting and standoff distance, both total depth of jet penetration, L_t, and depth at which the maximum width of the penetration pattern occurred, L_m, decreased progressively. For a standoff distance of zero, the maximum width of the penetration pattern, L_w, increased progressively with increasing viscosity at each of the injection settings. Conversely, when the standoff distance was 2.5 mm, L_w decreased progressively with increasing viscosity. Studies with neonate porcine skin revealed that an injection protocol comprising an 8.98 mPas solution, an arbitrary injection setting of 8 and a standoff distance of zero was capable of delivering photosensitisers to a volume of tissue (L_t of 2.91 mm, L_m of 2.14 mm, L_w of 5.10 mm) comparable to that occupied by a typical nodular basal cell carcinoma. Both ALA and TMP were successfully delivered using jet injection, with peak tissue concentrations (67.3 mg cm⁻³ and 5.6 mg cm⁻³, respectively) achieved at a depth of around 1.0 mm and substantial reductions in drug concentration seen at depths below 3.0 mm. Consequently, jet injection may be suitable for selective targeting of ALA or preformed photosensitisers to skin tumours.
Photodynamic inhibition of enzymatic detachment of human cancer cells from a substratum
2004, Biochimica et Biophysica Acta - General Subjects
Photodynamic therapy (PDT) is currently used for cancer treatment. It is shown that sublethal PDT of human WiDr adenocarcinoma cells and D54Mg glioblastoma cells with 5-aminolevulinic acid (ALA), disulfonated tetraphenylporphyrine (TPPS_2a), or MitoTracker Red (MTR) inhibits their trypsin-induced detachment from a plastic substratum. TPPS_2a was bound selectively to the plasma membrane, whereas MTR was found in mitochondria. Both granular and diffuse fluorescence of ALA-derived protoporphyrin IX (PpIX) was observed in the perinuclear cytoplasm but not in the plasma membrane of WiDr cells stained for 2 h with 1 mM ALA. In D54Mg cells, PpIX fluorescence was observed not only in the cytoplasm but also in the plasma membrane. Fluorescence measurements showed a progressive accumulation of PpIX in the WiDr cells during incubation with ALA and a PpIX efflux into the medium after 1 h or longer incubation. PpIX retained in the plasma membrane during efflux may be responsible for PDT-induced impairment of cell adhesion. On the other hand, MTR-PDT or ALA-PDT after 15-min incubation, when the newly synthesized PpIX should remain in mitochondria, also inhibited enzymatic cell detachment. Therefore, photodynamic targeting of mitochondria, remote from the cell surface where adhesion occurs, may disturb cell adhesion. Photodynamic inhibition of enzymatic cell detachment may be related to PDT-induced inhibition of tumour metastasis.

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In-vitro investigation of ALA-induced protopoyphyrin IX

Abstract

J. Photochem. Photobiol. B: Biol.

Biochem. Biophys. Acta

Int. J. Biochem.

J. Photochem. Photobiol. B: Biol.

J. Photochem. Photobiol. B: Biol.

J. Photochem. Photobiol. B: Biol.

Cancer Lett.

J. Photochem. Photobiol. B: Biol.

Clinical use of endogenous protoporphyrin IX in photodynamic therapy, B.o.A.

Synergistischer Effekt von d-Aminolävulisäure (ALA) and photodynamischer Lasertherapie anhand eines in vitro-Modells mit dem ATP-Chemosensitivitätsassay

Lasermedizin

Photosensibilisierung humaner Urothelkarzinomzellen durch 5-Aminolävulinsäure

Lasermedizin

In-vitro-Untersuchungen zur Aufnahme von 5-ALA and Retention von Protoporphyrin IX

Lasermedizin

ALA zur Photodynamischen Therapie an zwei Zell-Linien

Lasermedizin