Study of the efficacy of 5 ALA-mediated photodynamic therapy on human larynx squamous cell carcinoma (Hep2c) cell line

5-aminolevulanic acid (ALA), a precursor of Protoporphyrin IX, was evaluated as an inducer of photodamage on Hep2c, human larynx squamous cell carcinoma, cell line. Porphyrins are used as active cytotoxic antitumor agents in photodynamic therapy (PDT). The present study evaluates the effects of photodynamic therapy (PDT) with 5-aminolevulinic acid (5-ALA) using human larynx cells as experimental model. Hep2c cell line was irradiated with red light (a diode laser, λ = 635 nm). The influence of different incubation times and concentrations of 5-ALA, different irradiation doses and various combinations of photosensitizer and light doses on the cellular viability of Hep2c cells were studied. The optimal uptake of photosensitizer ALA in Hep-2c cells was investigated by means of spectrometric measurement. Cells viability was determined by means of neutral red assay (NR). It was observed that sensitizer or light doses have no significant effect on cells viability when studied independently. The spectrometric measurements showed that the maximal cellular uptake of 5-ALA occurred after 7 h in vitro incubation. The photocytotoxic assay showed that light dose of 85 J/cm² gives effective PDT outcome for Hep2c cell line incubated with 55 μg/ml of 5-ALA with a conclusion that Hep2c cell line is sensitive to ALA-mediated PDT.     

In vitro study of cell death with 5-aminolevulinic acid based photodynamic therapy to improve the efficiency of cancer treatment

Photodynamic therapy (PDT) is a kind of photochemo therapeutic treatment that exerts its effect mainly through the induction of cell death. Distinct types of cell death may be elicited by different PDT regimes. In this study, efforts are underway to optimize PDT protocols for improved efficacy and combination of all three PDT mechanisms involved in the different human carcinomas cell narcosis. Our in vitro cell culture experiments with 5-aminolevulanic acid (ALA) a clinically approved photiosensitizer (PS) and 635 nm laser light have yielded promising results, as follow: (1) (human cervical cancer (HeLa) cell line incubated, for 18 h, with 30 μg/ml of 5-ALA, treated with laser light dose of 50 J/cm² can produce 85% of cell killing (2) human larynx carcinoma (Hep2c) cell line incubated, for 7 h, with 55 μg/ml of 5-ALA, treated with laser light dose of 85 J/cm² can produce 75% of cell killing (3) human liver cancer (HepG2) cell line incubated, for 22–48 h, with 262 μg/ml of 5-ALA, treated with laser light dose of 120 J/cm² can produce 95% of cell killing (4) human muscle cancer (RD) cell line incubated, for 47 h, with 250 μg/ml of 5-ALA, treated with laser light dose of 80 J/cm² can produce 76% of cell killing (5) Human embryonic kidney (HEK293T) cell line incu-bated, for 18 h, with 400 μg/ml of 5-ALA, treated with laser light dose of 40 J/cm2 can produce 82% of cell killing confirming the efficacy of photodynamic therapy.     

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.     

Inactivation of bovine immunodeficiency virus by photodynamic therapy with HMME

To investigate the effect of photodynamic therapy (PDT) with hematoporphrin monomethyl ether (HMME) on bovine immunodeficiency virus (BIV) can provide the basis theory for photoinactivation of human immunodeficiency virus (HIV). To assess the protection of HMME-PDT on the cell line Cf2Th infected with BIVR29 by 3-(4,5)-dimethylthiahiazol-2-yl-3,5-di-phenytetrazolium bromide (MTT) with power density of 5 and 25 mW/cm2 and energy density from 0.6 to 3 J/cm<'2>. To observe the inhibition of membrane fusion using a new reporter cell line BIVE by fluorescence microscope. HMME-PDT has significant protectant effects on Cf2Th-BIVR29 with both power densities, especially in the group of high power density. Fluorescent microscope shows that there is no significant difference between the group of PDT and control, which means PDT could not inhibit the BIV-mediated membrane fusion.     

新型光动力学疗法光敏剂5-ALA-GNPs的制备及其光谱分析

通过聚二烯丙基二甲基胺盐酸盐和氯金酸制备阳离子纳米金,将纳米金和5-氨基乙酰丙酸(5-aminolevulinic acid, 5-ALA)通过静电吸附作用有效结合得到新型光敏剂。应用共振瑞利散射光谱, 紫外-可见吸收光谱, 透射电镜和激光散射等方法对其进行了表征。结果表明通过这种方法纳米金与5-ALA可以有效结合。这种新型光敏剂对提高光动力学疗法临床疗效具有重要指导意义。     

Holographic Study of the Response of HeLa Cells to Photodynamic Treatment with Endogenously Generated Protoporphyrin IX

Optics and Spectroscopy - Changes in morphological and optical parameters of HeLa cells preincubated with 5-aminolevulinic acid (5-ALA), after photodynamic treatment (PDTr) with different intensity...     

Photodynamic damage study of HeLa cell line using ALA

The present study evaluates the photodynamic damage with 5-aminolevulinic acid (5-ALA) using HeLa as experimental model. HeLa cell line was irradiated with red light (He-Ne laser, λ = 632.8 CW nm). The influence of different incubation times and concentrations of 5-ALA, different irradiation doses and various combinations of photosensitizer and light doses on the cellular viability of HeLa cells were studied. The optimal uptake of photosensitizer ALA in HeLa cells was investigated by means of PpIX fluorescence intensity by exciting the HeLa cell suspension at 450 nm and a detection wavelength set at 690 nm. Cells viability was determined by means of trypan blue solution. The spectrometric measurements showed that the maximal cellular uptake of 5-ALA occurred after 4 h in vitro incubation. We found that the combination with 5-ALA and laser irradiation leads to time/concentration-dependent increase of cells death and also energy doses-dependent enlarge the cells death. The fluorescence intensity after PDD of carcinoma cells reduce when compared with the control group. The fluorescence emission spectral profiles after PDD of carcinoma cells showed a dip around 425–525 nm when compared with the control group. This may be due to the damage of mitochondria component of cells. The percentage of HeLa cells after PDD shows that the percentage of cells survival rate as function of laser dose (power). Hence it is clear that at 200 μg/ml ALA and 20 mW laser irradiation, more than 70% of HeLa cells were dead after 15 min.     

Serial MR imaging of intracranial metastases after radiosurgery

Purpose: To evaluate the spatiotemporal evolution of radiosurgical induced changes both in metastases and in normal brain tissue adjacent to the lesions by serial magnetic resonance (MR) imaging. Methods and Materials: Thirty-five intracranial metastases of different primaries were treated in 25 patients by single high-dose radiosurgery. MR images acquired before radiosurgery were available in all patients. Sixty-three follow-up MR studies were performed in these patients including T₂- and contrast-enhanced T₁-weighted MR images. The average follow-up time was 9 ± 5 months (mean ± standard deviation [SD]). Based on contrast-enhanced T₁-weighted MR images, tumor response was radiologically classified in the following four groups: stable disease was assumed if the average tumor diameter after treatment did not show a tumor shrinkage of more than 50% and an increase of more than 25%, partial remission as a shrinkage of tumor size of more than 50%, a disappearance of contrast-enhancing tumor as a complete remission, and an increase of tumor diameter of more than 25% as tumor progress. Moreover, we analysed signal changes on T₂-weighted images in brain parenchyma adjacent to the enhancing metastases. Results: The overall mean survival time was 10.5 ± 7 months, with a 1-year actuarial survival rate of 40%. Stable disease, partial or complete remission of the metastatic tumor was observed in 22 patients (88%). Central or homogeneous loss of contrast enhancement appeared to be a good prognostic sign for stable disease or partial remission. This association was statistically significant (p < 0.05). Three patients (12%) suffered from tumor progression. In eight patients (32%) with stable disease or partial remission, signal changes on T₂-weighted images were observed in tissue adjacent to the contrast enhancing lesions. A progression of the high signal on T₂-weighted images was seen in seven of the eight patients between 3 and 6 months after therapy, followed by a signal regression 6–18 months after irradiation. Conclusion: MR imaging is a sensitive imaging tool to evaluate tumor response as well as the presence or absence of adjacent parenchymal changes following radiosurgery. Loss of homogeneous or central contrast enhancement on Gd-enhanced MR images appeared to be a good prognostic sign for tumor response. Tumor shrinkage seems not to be dependent on time. In addition, most cases of radiation induced changes in normal brain parenchyma observed on T₂-weighted images seem to be self limited.     

Correlation of cytotoxicity and depth of necrosis of the photoproducts of photogem®

Photodynamic therapy (PDT) is an approved modality for cancer treatment, which involves the administration of a photosensitive drug (PS) that is selectively accumulated in neoplastic tissues and their vasculature and subsequently can be activated with light at the appropriate wavelength to generate reactive molecular species that are toxic to tissues. In PDT, a great part of the used PS suffers degradation by light (photobleaching) that involves a decrease in the absorption and intensity of fluorescence of the photosensitizer as well as photoproduct formation evidenced by the appearance of a new absorption band. In this study, we investigated the correlation of cytotoxicity and depth of necrosis of Photogem and its photoproducts obtained previously by irradiation at 514 and 630 nm. The cytotoxicity for degraded Photogem decreases with the previous irradiation time of Photogem solution suggesting that the photoproducts of Photogem are less cytotoxics than the original formulation. A transition between the necrosed epithelium and healthy epithelium of normal liver of rats after irradiation at 630 nm was observed with irradiated and nonirradiated PS. It is observed that the depth of necrosis only at irradiation dose of 150 J/cm² in both concentrations is greater for Photogem followed by Photogem degradated previously at 514 and then at 630 nm. The results obtained suggest that the threshold of necrosis values is lower for Photogem followed by its photoproducts formed, suggesting that the photoproducts present a low photodynamic activity. If the photosensitizer degradation happens at the same time as tumor destruction, the drug degradation can be complete before reaching the threshold of necrosis; then it is very important to control the drug concentration and light intensity of irradiation during PDT.     

Effectiveness of Photogem® activated by LED on the decontamination of artificial carious bovine dentin

The aim of this study was the evaluation of the effectiveness of photodynamic therapy on the decontamination of artificially induced carious bovine dentin, using Photogem® as the photosensitizer agent and an LED device as a light source. Dentin samples obtained from bovine incisors were immersed in sterile broth supplemented by Lactobacillus acidophillus 10⁸ colony formation units (CFU) and Streptococcus mutans 10⁸ CFU. Different concentrations of photosensitizer, PA = 1 mg/ml, PB = 2 mg/ml, and PC = 3 mg/ml, and two fluences, D = 24 J/cm² and D = 48 J/cm², were investigated. After CFU counting per milligram of carious dentin and statistical analysis, we observed that the photodynamic therapy (PDT) parameters used were effective for bacterial reduction in the in vitro model under study. The best result was achieved with the application of Photogem® at 2 mg/ml and photoactivated under 24 J/cm² showing a survival factor of 0.14. At higher photosensitizer concentrations, a higher dark toxicity was observed. We propose a simple mathematical expression for the determination of PDT parameters of photosensitizer concentration and light fluence for different survival factor values. Since LED devices are simpler and cheaper compared to laser systems, it would be interesting to verify their efficacy as a light source in photodynamic therapy for the decontamination of carious dentin.     

Can efficiency of the photosensitizer be predicted by its photostability in solution?

We have investigated a possible correlation between the photostability and photodynamic efficacy for different photosensitizers; hematoporphyrin derivatives and chlorines. To perform such analysis, we combined the depth of necrosis (d _nec) measurement, expressed by the light threshold dose and a photodegradation parameter, measured from investigation of photosensitizer degradation in solution. The d _nec analysis allows us to determine the light threshold dose and compare its value with the existent results in the literature. The use of simple models to understand basic features of Photodynamic Therapy (PDT) may contribute to the solid establishment of dosimetry in PDT, enhancing its use in the clinical management of cancers and others lesions. Using hematoporphyrin derivatives and chlorines photosensitizers we investigated their properties related to the photodegradation in solution and the light threshold dose (D _th) in rat livers.     

Investigation of photodynamic therapy on streptococcus mutans of oral biofilm

We investigated the effect of photodynamic therapy (PDT) with hematoporphyrin monomethyl ether (HMME) on the viability of Streptococcus mutans (S. mutans) cells on biofilms in vitro. Streptococcus mutans is the primary etiological agent of human dental caries. Since dental caries are localized infections, such plaque-related diseases would be well suited to PDT. The diode laser used in this study had the wavelength of 635 nm, whose output power was 10 mW and the energy density was 12.74 J/cm2. HMME was used as photosensitizer. Samples were prepared and divided into five groups: (1) HMME; (2) Laser; (3) HMME Laser; (4) Control group ( ) with chlorhexidine; and (5) Control group (-) with sterile physiological saline. Inoculum of S. mutans incubated with HMME also examined with fluorescence microscopy. PDT exhibited a significantly (P < 0.05) increased antimicrobial potential compared with 20 μm/mL HMME only, laser only, 0.05% chlorhexidine, and 0.9% sterile physiological saline, which reduced the S. mutans of the biofilm most effectively. Laser and 0.05% chlorhexidine were caused reduction in the viable counts of S. mutans significantly different (P < 0.05) also, but these two test treatments did not statistically differ from each other. HMME group did not statistically differ with negative control group. Fluorescence microscopy indicated that HMME localized primarily in the S. mutans of the biofilm. It was demonstrated that HMME-mediated PDT was efficient at killing S. mutans of biofilms and a useful approach in the treatment of dental plaque-related diseases.     

Photodynamic activity of different dyes

Photodynamic therapy (PDT) is a technique for inducing tissue damage with light irradiation of a drug selectively retained in malignant tissue. Many kinds of compounds are known with photosensitizing properties including dyes, drugs, cosmetics, chemicals, and many natural substances. There are different classes of sensitizers used for medical purposes such as tetrapyrroles (porphyrins and derivatives, chlorophyll, phylloerythrin, phthalocyanines), tricyclic dyes with different meso-atoms (acridine orange, proflavine, riboflavine, methylene blue, fluorescein, eosine, erythrosine, rose bengal), and furocoumarins (psoralen and its methoxyderivatives xanthotoxin, bergaptene). In this work, we performed one comparative cytotoxic study of the photodynamic activity presented by tricyclic dyes (methylene blue, fluorescein and erythrosine) and the commercial Russian photosensitizer Photogem® (hematoporphyrin derivative). For this purpose, three cell lines were used: HEp-2 (tumor cells), VERO and McCoy (nontumor cells), and a yeast strain. The wavelength used for irradiation was 630 nm, the same as used in PDT for medical purposes, since it is in the therapeutic window, i.e., where light can penetrate more into the tissues. The results suggest that Photogem® is more cytotoxic and more photocytotoxic than the studied tricyclic dyes in nontumor and tumor cells. These dyes present less cytotoxicity (around half) in normal cells (dark and light) than in tumor cells. In the experiments with microorganisms, methylene blue presented a better photodynamic effect than Photogem®. These results can be explained by the fact that it is more difficult for Photogem® to penetrate in microorganism membranes than mammalian cell membranes. As for Photogem®, these tricycle dyes present a higher cytotoxicity in tumor cells. These data suggest that methylene blue can be an option in photodynamic inactivation as well as in photodynamic therapy, mainly for superficial lesions.     

Study of the efficacy of photofrin®-Mediated PDT on human hepatocellular carcinoma (HepG2) cell line

Laser Physics - The present study evaluates the effects of photodynamic therapy (PDT) with Photofrin® using human liver cancer cells (HepG2) as an experimental model. We optimized the...     

Combined laser and photodynamic treatment in extensive purulent wounds

Recently, photodynamic therapy (PDT) has been used for the treatment of festering wounds and trophic ulcers. An important advantage of PDT is its ability to affect bacterial cultures that are resistant to antibiotics. However the use of PDT alone does not usually guarantee a stable antiseptic effect and cannot prevent an external infection of wounds and burns. In this work attention is focused on the healing of the extensive soft tissues wounds with combined laser therapy (LT) and PDT treatment. At the first stage of this process festering tissues (for example spacious purulent wounds with area more than 100 cm²) were illuminated with high-energy laser beam (with power 20 W) in continues routine. The second stage involves “softer” PDT affect, which along with the completion stages of destruction pathological cells, stimulating the process of wound granulation and epithelization. Also, according to our previous results, photosensitizer (photoditazin) is introduced inside the wound with different amphiphilic polymers for increasing the PDT efficacy.     

Tunable wavelength laser medical system for treating oncological diseases

A new laser medical system (LMS) “LITT-FDT” for treating oncological diseases is presented. The laser system is based on a dye laser pumped by a copper vapor laser. The system features gradual wavelength tuning which allows operation almost with any photosensitizer (PS) when treating oncological diseases by the photodynamic therapy (PDT) method. The results of clinical trials of skin cancer treatment by the PDT method using the “LITT-FDT” laser system are considered.     

Light parameters influence cell viability in antifungal photodynamic therapy in a fluence and rate fluence-dependent manner

The aim of this study was to investigate the influence of light parameters on yeast cells. It has been proposed for many years that photodynamic therapy (PDT) can inactivate microbial cells. A number of photosensitizer and light sources were reported in different light parameters and in a range of dye concentrations. However, much more knowledge concerning the importance of fluence, fluence rate and exposure time are required for a better understanding of the photodynamic efficiency. Suspensions (10⁶ CFU/mL) of Candida albicans, Candida krusei, and Cryptococcus neoformans var. grubii were used. Two fluence rates, 100 and 300 mW/cm² were compared at 3, 6, and 9 min of irradiation, resulting fluences from 18 to 162 J/cm². The light source was a laser emitting at λ = 660 nm with output power adjusted at 30 and 90 mW. As photosensitizer, one hundred-μM methylene blue was used. Temperature was monitored to verify possible heat effect and reactive oxygen species (ROS) formation was evaluated. The same fluence in different fluence rates showed dissimilar levels of inactivation on yeast cells as well as in ROS formation. In addition, the increase of the fluence rate showed an improvement on cell photoinactivation. PDT was efficient against yeast cells (6 log reduction), and no significant temperature increase was observed. Fluence per se should not be used as an isolate parameter to compare photoinactivation effects on yeast cells. The higher fluence rate was more effective than the lower one. Furthermore, an adequate duration of light exposure cannot be discarded.     

In vivo relaxation time measurements on a murine tumor model—Prolongation of T1 after photodynamic therapy

RIF tumors implanted on mice feet were investigated for changes in relaxation times (T₁ and T₂) after photodynamic therapy (PDT). Photodynamic therapy was performed using Photofrin II as the photosensitizer and laser light at 630 nm. A home-built proton solenoid coil in the balanced configuration was used to accommodate the tumors, and the relaxation times were measured before, immediately after, and up to several hours after therapy. Several control experiments were performed using the untreated tumors, tumors treated with Photofrin II alone, or tumors treated with laser light alone. Significant increases in T₁s of water protons were observed after PDT treatment. In all experiments, ³¹P spectra were recorded before and after the therapy to study the tumor status and to confirm the onset of PDT. These studies show significant prolongation of T₁s after the PDT treatment. The spin-spin relaxation measurements, on the other hand, did not show such prolongation in T₂ values after PDT treatment.     

Photosensitizer Targeting: Mitochondrion-Targeted Photosensitizer Enhances Mitochondrial Reactive Oxygen Species and Mitochondrial Calcium-Mediated Apoptosis

Using laser scanning imaging microscopy, our previous studies have demonstrated visible laser irradiation induced significant generation of mitochondrial reactive oxygen species (mROS) and apoptosis. In addition, photodynamic therapy (PDT) with a mitochondrion-targeted photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA), significantly enhanced visible laser irradiation induced mitochondrial dysfunction and apoptosis. To validate the critical role of the mitochondrion as well as how mitochondrial mechanisms were involved in the PDT of BPD-MA, subcellular PDT with BPD-MA were performed using region irradiation and recorded by multi-photon imaging microscopy coupled with mitochondrial specific fluorescent probes in single live C6 glioma cells. Specifically, mROS and mitochondrial free calcium (mCa²⁺) were simultaneously measured from cells received subcellular PDT with BPD-MA in different compartments including the mitochondrion, the cytosol and the nucleus. Results revealed that significant formation of mROS accompanied by elevation of mCa²⁺ was found only in the mitochondrial PDT with BPD-MA. Subsequently, mitochondrial PDT with BPD-MA resulted in severe mitochondrial swelling, plasma membrane blebing and apoptotic death. We conclude that via augmented mROS formation and mCa²⁺ elevation PDT with a mitochondrion-targeted photosensitizer extensively enhances mitochondria-mediated apoptosis and, thus, may provide high eradication efficacy for clinical cancer treatment.     

Polydopamine Nanoparticle as a Multifunctional Nanocarrier for Combined Radiophotodynamic Therapy of Cancer

Combination of different therapeutic strategies to treat cancer has attracted tremendous attention in recent years. Herein, the authors develop polydopamine (PDA) nanoparticles with polyethylene glycol (PEG) modification as a multifunctional nanocarrier for coloading photosensitizer chlorine6 (Ce6) and curcumin (Cur) for combined photodynamic therapy (PDT) and radiotherapy (RT) of cancer. PEGylated PDA nanoparticles (PDA‐PEG) exhibit well water soluble and biocompatible in different physiological solutions and cause no obvious toxicity to cancer cells. In this nanoparticle, the loaded Ce6 can trigger the generation of single oxygen under near‐infrared laser irradiation for PDT, while the loaded Cur can act as an excellent radiosensitizer under X‐ray irradiation for enhanced external RT. As demonstrated by in vitro and in vivo therapeutic efficiency, combined PDT and RT based on PDA‐PEG/Cur/Ce6 nanoparticles exhibits significant inhibition the growth of cancer cells, revealing perfect performance in cancer treatment. Therefore, the study not only presents a polymer‐based theranostic platform for cancer treatment but also demonstrates the potential applications of combined RT and PDT for the future clinic cancer therapy.