Photodynamic therapy of experimental tumours with Zn(II)-phthalocyanine and pulsed laser irradiation

The effectiveness of a pulsed dye laser (673 nm) for photodynamic therapy (PDT) of tumours in the presence of Zn(II)-phthalocyanine (ZnPc) was evaluated using Lewis lung carcinoma-bearing mice. The tumours were irradiated with different pulse energies (from 0.4 to 10 mJ) at a constant fluence of 0.6 J cm^–2 at 24 h after administration of 0.25 mg kg^–1 body weight liposome-incorporated ZnPc. Maximal PDT effect, as evaluated by changes in mean tumour diameter, animal survival time and histological evaluation of tumour necrosis, was observed after 3.0 mJ pulse energy irradiation which appears to yield a deeper light penetration and a more efficient sensitizer excitation when compared with lower or higher pulse energies. Electron microscopic analysis of photo-treated tumour indicates preferential damage to malignant tissue as compared to endothelial cells.     

Adverse effects reported in pulsed dye laser treatment for port wine stains

A retrospective study was conducted over a 762-day period to investigate the incidence of adverse effects following pulsed dye laser treatment for port wine stains using the Chromos 585 nm pulsed dye laser (PDL). Treatment energy densities typically ranged from 4.0 J/cm² to 7.5 J/cm². Treatment was predominantly delivered as a 7 mm-diameter pulse of laser light, although a 5 mm pulse was occasionally used. Nine patients experienced one adverse event each, with scabbing, blistering (prolonged in one case that persisted for more than 2 months), hypopigmentation or hyperpigmentation; one of the nine patients developed both blistering and hypopigmentation. The incidence of adverse effects per treatment was 1.4% (1.7% for adults and 0.6% for children). The lower leg was the most common site for an adverse effect; four out of 22 patients (18%) receiving PDL treatment for lower leg port wine stains (PWSs) suffered an adverse effect (three cases of hyperpigmentation and one of hypopigmentation and blistering). Four patients suffered an adverse effect following an increase in the energy density of the laser treatment. There was no statistically significant correlation between laser performance and the occurrence of adverse effects.     

Photosensitized patients and operating lights

Photosensitized patients who receive intra-operative photodynamic therapy are subjected to the intense brightness of operating lights. In order to investigate the potential phototoxic effects of this therapy, light energy densities were measured in non-photosensitized patients undergoing major abdominal surgery. The results show energy densities of 0.14 to 12.51 J cm^–2 of polychromatic light for different operative fields. By a consideration of photon absorption numbers, these levels have a photodynamic equivalent of 0.34 to 30.06 J cm^–2 of 630 nm light at the surface of tissue. If surgeons are unaware of the possible phototoxic effects of operating lights, some tissues could receive an unacceptably high energy density of polychromatic light. However, operating lights should not give significant phototoxic effects if the surgeon is aware and takes simple precautions.     

上转换纳米粒子介导的光动力疗法杀伤脊髓星形胶质细胞的实验研究

 目的 探讨上转换纳米粒子（upconversion nanoparticles, UCNPs）介导的光动力疗法对大鼠脊髓星形胶质细胞的体外杀伤效应。方法 体外无菌条件下取新生SD大鼠脊髓组织,采用机械吹打及胰酶消化法制成细胞悬液,通过差速贴壁和恒温摇床震荡去除杂细胞,利用GFAP免疫荧光染色鉴定,成功分离培养大鼠脊髓星形胶质细胞。采用MTT法检测不同浓度UCNPs-MC540[以上转换纳米为载体和能量转换器,并使用偶联光敏剂部花青540（Merocyanine 540）构建的复合物]、不同能量980 nm激光照射对脊髓星形胶质细胞活力的影响以及在980 nm近红外激光照射下UCNPs-MC540介导的光动力疗法的细胞杀伤效应;应用浓度为200 μg/ml的 UCNPs-MC540培养液与星形胶质细胞共培养12 h后,给予2 000 J/cm²的980 nm近红外激光进行照射,以同样条件培养而未作激光照射光动力治疗的细胞作为对照组;分别制作透射电镜标本,透射电子显微镜下观察细胞形态改变。结果 不同浓度UCNPs-MC540及不同照射剂量980 nm激光照射处理后细胞存活率的差异无统计学意义。在浓度100 μg/ml的UCNPs-MC540作用细胞12 h后分别给予不同能量激光照射,细胞存活率随着激光剂量的增加而下降,差异有统计学意义。不同浓度的UCNPs-MC540作用于细胞12 h后予2 000 J/cm²能量激光照射,随着UCNPs-540浓度的增加,细胞的存活率降低,差异有统计学意义。与未作激光照射光动力治疗细胞的对照组比较,细胞与浓度为200 μg/ml的UCNPs-MC540共培养,能量为2 000 J/cm²激光照射后,透射电镜检查显示细胞呈凋亡表现,形成具有特征性的凋亡小体。结论 由上转换荧光纳米NaYF:Er,Yb与光敏剂MC540连接的纳米复合物UCNPs-MC540介导的光动力疗法对星形胶质细胞有良好的杀伤效果,其机制可能为诱导细胞发生凋亡,为脊髓损伤的治疗提供新的思路,扩大光动力疗法的研究应用范围。     

Development of an alternative light source to lasers for photodynamic therapy: 2. Comparative in vivo tumour response characteristics

The performance of a low cost, table-top/portable light source was tested against an argon ion pumped dye laser for in vivo photodynamic therapy (PDT). The prototype delivers up to 1 W via a 4 mm flexible lightguide within a 30 nm bandwidth centred at any wavelength from 300 nm to 1200 nm at fluence rates of up to 8 W cm^–2. An in situ bioassay using regrowth delay of tumour T50/80 was used to quantify the relative efficacy of the prototype with a laser. The tumours were sensitized with haematoporphyrin derivative (HpD) and externally irradiated. There was no significant difference in the response of the tumour to treatment between the two light sources (p = 0.69). Mean growth delays ranged from 2 days (light dose 10 J cm^–2) to 20 days (light dose 100 J cm^–2). The estimate for the difference in means (laser minus prototype growth delay) was only 0.66 days and was not statistically significant. This in vivo study demonstrates that the prototype is equivalent to a laser in PDT effect. The device has low capital/running cost, is simple to use and is one of the most powerful, spectrally efficient non-laser PDT sources available.     

In vitro behavior of Porfimer sodium and Talaporfin sodium with high intensity pulsed irradiation

We studied pulse energy density dependence of two distinctive clinical photosensitizers, Porfimer sodium and Talaporfin sodium, in terms of oxygen consumption, photodegradation in these photosensitizer solutions, and rat prostate cancer cell line photocytotoxicity. The transient transmittances during the pulsed irradiation to these photosensitizer solutions were measured with the pulse energy densities ranging from 0.31 to 31 mJ/cm². We revealed that Talaporfin sodium was easier to produce absorption saturation than Porfimer sodium. The significant suppression of Talaporfin sodium mediated oxygen consumption, photodegradation, and photocytotoxicity which were observed with pulse energy densities increasing from 0.5 to 10 mJ/cm². This result could be mainly attributed to absorption saturation. On the other hand, Porfimer sodium did not display significant absorption saturation with the pulse energy densities ranging from 0.31 to 31 mJ/cm². The photodegradation mechanism for Porfimer sodium changed at high pulse energy density. This phenomenon might accelerate the photodegradation and cause the photocytotoxicity suppression.     

Endoscopic photodynamic therapy in lung cancer

The main purpose of cancer therapy is to treat malignant tissue with the least damage to normal surrounding structures. Photodynamic therapy (PDT) seems to be able to fulfil this simple but fundamental premise.The mechanism of action of the photosensitizer—light system can be summarized in two main points. Chiefly, it seems to be a photodynamic process, with energy transfer from the light to the photosensitizer and from it to the oxygen molecules. Oxygen is excited and becomes singlet oxygen, which is extremely reactive and very noxious for tissues in which it develops. Secondly, a thermal mechanism related to light absorption and consequent temperature rise also seems to be involved in malignant necrosis by PDT.Thirteen males were submitted to endoscopic PDT. A total of 15 treatments were given: 2 patients were submitted to 2 sessions of PDT. Forty-eight hours after HPD administration (72 h in a few cases), the lesions were exposed to a 630 nm light from an argon-dye laser system.The total estimated energy dose delivered to the tumour surface was 90–150 J/cm² in 11 cases. All cases treated responded well and total disappearance was obtained. Median follow-up was 9.5 months (1–20 months) and the estimated energy delivered from 90–600 J/cm². No major complications were reported.     

Comparison of the photodynamic fungicidal efficacy of methylene blue, toluidine blue, malachite green and low-power laser irradiation alone against Candida albicans

This study was to evaluate specific effects of photodynamic therapy (energy density 15.8 J/cm², 26.3 J/cm² and 39.5 J/cm²) using methylene blue, toluidine blue and malachite green as photosensitizers and low-power laser irradiation on the viability of Candida albicans. Suspensions of C. albicans containing 10⁶ cells/ml were standardized in a spectrophotometer. For each dye, 120 assays, divided into four groups according to the following experimental conditions, were carried out: laser irradiation in the presence of the photosensitizer; laser irradiation only; treatment with the photosensitizer only; no exposure to laser light or photosensitizer. Next, serial dilutions were prepared and seeded onto Sabouraud dextrose agar for the determination of the number of colony-forming units per milliliter (CFU/ml). The results were subjected to analysis of variance and the Tukey test (P < 0.05). Photodynamic therapy using the photosensitizers tested was effective in reducing the number of C. albicans.. The number of CFU/ml was reduced by between 0.54 log₁₀ and 3.07 log₁₀ and depended on the laser energy density used. Toluidine blue, methylene blue and malachite green were effective photosensitizers in antimicrobial photodynamic therapy against C. albicans, as was low-power laser irradiation alone.     

Effect of infra-red low-power laser irradiation on regeneration of myelinated axons

The purpose of the present study was to examine the regeneration of myelinated axons under the effect of laser irradiation at various wavelengths and energy densities. Laser irradiation at 890 nm with an energy dose of 0.33 J cm^–2 as well as He-Ne laser irradiation failed to change the number of regenerating myelinated axons in distal nerve stumps on the 30th day after cutting the nerve. An increase of dose delivered to the skin to 9.33 J cm^–2 resulted in a 49% decrease in the number of myelinated axons. A 24% suppression of nerve regeneration was also registered using 1220 nm wavelength laser (dose 0.98 J cm^–2). This phenomenon is likely to be attributed to the stimulating effect of laser irradiation of the near-infra-red range on proliferation of fibroblasts and scar formation. 1220 nm of 7.2 J cm^–2 dose effected neither the growth nor the myelinization of axons in distal nerve stumps on the 20th day following nerve damage.     

Effect of Helium-Neon (He-Ne) Laser Irradiation on Dog Neoplasm Cells in Culture

The effects of laser light on the cellular proliferation have been extensively characterised. Low-power laser sources, such as the helium–neon (He-Ne) laser irradiation with a wavelength of 632.8 nm, have been found to produce photobiological and photodamaging effects with evidence of interference with cell proliferation functions. The present study has investigated the in vitro effect of He-Ne laser irradiation on the proliferative action of dog tumour cells in culture. Dose–response studies showed that repeated He-Ne irradiation (irradiance 12.8 mW/cm²) once a day for 4 consecutive days in a dose range between 0.13 and 2.08 J/cm² significantly increased with increasing energy density up to a laser dose of 0.26 J/cm², whereas at >1.04 J/cm², the cell proliferation decreased with increasing energy densities. It is concluded that the application of He-Ne laser irradiation at energy densities ranging from 0.13 J/cm² to 2.08 J/cm² produced different effects on cell proliferation in dog tumour cells in culture. Paper received for publication 27 June 1997; accepted following revision 6 February 1998.     

Experimental Cholelitholysis with the Pulsed Tunable Dye Laser

This study evaluates the pulsed tunable dye laser with wavelength 504 nm, frequency 10 Hz, and pulse width 1.2 μs for cholelitholysis. Power of 10–40 kW was directed through a 250-pm quartz fiber optic to ablate 55 gallstones (removed from 14 patients). The fiber was positioned in direct contact with the stones under saline. Power delivery was begun at 10 kW and increased in 10-kW increments until litholysis began. The range of power and energy necessary to fragment the gallstones was evaluated on four common bile ducts fresh autopsy specimens). Following fragmentation, all stones were analyzed. There were 35 cholesterol stones (3 calcified) and 20 bilirubin stones (4 calcified). Size ranged from 0.012 to 7.56 cm³ (mean 0.96 ± 1.41 cm³). Energy necessary for fragmentation ranged from 0.4 to 11.2 J (exposure time 1.0–28 s). Power necessary for fragmentation was 20 kW for 2/55 stones and 40 kW for 53/55 stones. At 40 kW (40 mJ/pulse), common bile duct perforation occurred within 1.1 ± 0.1 s (0.44 ± 0.04 J). The pulsed tunable dye laser can fragment gallstones of all compositions. The threshold for fragmentation is 40 kW, but common bile duct perforation occurs at this power. We conclude that laser radiation sufficient to fragment gallstones can injure the common bile duct.     

The relevance of pulse repetition rate and radiant exposure to the neurophysiological effects of low-intensity laser (820 nm/pulsed wave) irradiation upon skin temperature and antidromic conduction latencies in the human median nerve

The effects of low-intensity near-infra-red laser irradiation (820 nm; 1.5 and 9.0 J cm^–2; pulsed at 12 Hz, 73 Hz and 5 kHz) upon peripheral neurophysiology and skin temperature were investigated using antidromic conduction studies in the human median nerve in vivo. Healthy human volunteers (n = 90) were recruited and allocated randomly to either a control group (n=10) or one of eight experimental groups (two radiant exposures, 1.5 J cm^–2 and 9.0 J cm^–2 at one of three pulse repetition rates, 12 Hz, 73 Hz or 5 kHz, in addition to a placebo group for each radiant exposure;n = 10 all groups). Analysis of variance (ANOVA) demonstrated a significant (p0.05) decrease in skin temperature following irradiation at the lowest radiant exposure (1.5 J cm^–2) combined with pulse repetition rates of 73 Hz and 5 kHz, with the greatest effect at 73 Hz. These changes in skin temperature were coupled with increases in negative peak latency (NPL); ie changes in NPL were inversely related to changes in skin temperature. However, in contrast to the authors' previous findings using continuous wave (CW) laser irradiation, differences in NPL were not found to be significant. These findings, therefore, provide little evidence of the neuro-physiological effects of low-intensity infra-red irradiation at the dosage levels and pulse repetition rates used here.     

Catheter associated problems of multi-fibre systems in clinical excimer laser angioplasty

We report our experience in 60 patients using a recently designed 4 French ring catheter system for coronary and a 7 French ring catheter for peripheral percutaneous excimer laser angioplasty. The advantages in comparison to bare fibres comprise an improved flexibility and a central channel for insertion of guide wires to minimize the risk of mechanical vessel wall perforation. The peripheral laser catheter initially transmitted overall energies of 20 mJ pulse^–1 (51 mJ pulse^–1 mm^–2), the coronary device 4.5–5 mJ pulse^–1 (32 mJ pulse^–1 mm^–2). A 50% or more decrease of energy transmission was found in 43% of coronary catheters due to a number of blinded fibres with other fibres remaining intact. This was either due to a retrograde expansion of shock waves generated by the excimer pulse at a calcified lesion, or the result of a deleterious back-reflection of the laser light by contrast media. However, using both laser catheters ablation of plaque in vivo proved to be possible. Small mechanical defects of the catheter tips in 27% did not result in patient related complications. Our initial experience favours further improvement of ring catheters for percutaneous excimer laser angioplasty.     

Effects of an Er:YAG laser on mitochondrial activity of human osteosarcoma–derived osteoblasts in vitro

The aim of the present study was to investigate the collateral damage of an Er:YAG laser on bone cells in vitro using a special application tip designed for treatment of periimplantitis. Before laser irradiation, SaOs-2 osteoblasts (2×10⁴ cells) were inoculated into 96-well tissue culture plates and incubated for 48 h under standardised conditions. A total of 120 cell cultures were irradiated with an Er:YAG laser using a cone-shaped quartz glass fibre tip at energy settings of 40, 60, 80 and 100 mJ at 10 Hz (energy densities of 5.08, 7.62, 10.16 and 12.7 J cm^–2) for 10 s. Each energy setting was used at a distance of 1, 2 and 3 mm between the application tip and the bottom of the culture plate. Following irradiation, mitochondrial activity of the cells was measured using a luminescent cell viability assay. After laser irradiation, mitochondrial activity of SaOs-2 osteoblasts was significantly reduced when compared with nonirradiated cells (P<0.001), dependent on the energy setting used and the distance between the application tip and the bottom of the culture plate. Mitochondrial activity increased significantly with decreasing energy settings and increasing distances (P<0.001). Within the limits of the present study, it was concluded that an Er:YAG laser, used with a cone-shaped glass fibre tip designed for treatment of periimplantitis, has detrimental effects on mitochondrial activity of SaOs-2 osteoblasts in vitro at energy settings of 40, 60, 80 and 100 mJ (10 Hz).     

Thermal damage of blood vessels in a rat skin-flap window chamber using indocyanine green and a pulsed alexandrite laser: A feasibility study

The design criteria and feasibility of specifically targeting blood vessels for thermal damage by using a pulsed alexandrite infra-red laser to heat an intravascularly injected infra-redabsorbing dye, namely indocyanine green (ICG), is demonstrated. Theoretical calculations map the distribution of light and heat in and around the subcutaneous blood vessels in a rat skin-flap window chamber as functions of dye concentration, vessel size, and vessel depth. Theoretical calculations showed that an injected dose of 6–24 mgkg⁻¹ of ICG and a 120-μs, 1-J cm⁻² alexandrite laser pulse at a wavelength of 785 nm would be sufficient to achieve selective vascular damage to a depth of at least 0.15 cm. Feasibility experiments were performed which illustrated that an irradiation of 1.27 J cm⁻² of skin flaps in uninjected control rats showed no evidence of vascular damage while vascular damage was seen in skin flaps using an experimental protocol of 12 mg kg⁻¹ i.v. of ICG and an energy fluence of 0.76 J cm⁻². This procedure could conceivably prove useful in the treatment of vascular lesions or cancer.     

Interstitial photodynamic therapy in the Dunning R3327-AT6 prostatic carcinoma

Interstitial photodynamic therapy (PDT) could be an alternative radical treatment for prostate cancer. The ability to predict the depth of necrosis is necessary for light treatment planning using multiple optical fibres. The extent of PDT necrosis was studied in subcutaneously implanted R3327-AT6 Dunning prostate tumours which had similar optical characteristics to human prostate. Tumour-bearing subjects were given 20 mg kg^–1 Haematoporphyrin esters (HPE) and irradiated 24 h later with 630 nm laser light. Five subjects per group were treated with increasing light doses (50–450 J cm^–1) delivered interstitially via a single 2 cm long cylindrical diffuser. After 450 J cm^–1 of irradiation, 4.3±0.8 cm³ [standard error of the mean (s.e.m.)] of tumour tissue was necrosed to a depth of 10.5±0.8 mm around the diffuser. There was an approximately linear correlation between the volume of PDT necrosis around the fibre and prescribed light dose. The mean threshold light dose for PDT effect was 18±2 J cm^–2. In this tumour with a mean photosensitizer concentration of 16±1.5g g^–1, low light doses produced tumour necrosis. PDT using multiple diffusers could destroy a relatively large tumour volume and the diffusion theory model reliably predicted the depth of necrosis.     

Experimental photoablation of meniscus cartilage by excimer laser energy

Summary Excimer lasers have been demonstrated to provide a very precise and circumscribed ablation of synthetic polymers and biological tissues. We investigated in vitro the use of ultrashort pulsed ultraviolet excimer-laser energy for controlled removal of meniscus cartilage under the aspects of arthroscopic meniscectomy. A krypton-fluorine gas mixture was used to achieve laser emission of 248-nm wavelength. A total of 22 human menisci obtained either by operation or necropsy were irradiated over a range of energy fluence (2.15–3.07 J/cm²/pulse), repetition rates (5–20 Hz), and exposure time (15–60 s). Ablation rates of 4.00–5.76 m per pulse were obtained. Light-microscopic examinations demonstrated tissue ablation without any evidence of pathological changes associated with continuous-wave laser irradiation. Effects of laser energy were clearly limited to the target of the laser beam, and tissue removal proceeded without production of heat or smoke. Due to the lack of pathological alterations observed, excimer-laser irradiation of meniscus cartilage may prove to be advantageous for precisely cutting and removing menisci without injury to the surrounding normal tissue. Clinical application of excimer-laser irradiation includes the development of suitable fiberoptics and laser coupling, as well as modification of fiber tips.Funds were received from theFonds zur Förderung der wissenschaftlichen Forschung (Ministry of Science and Research, Austria)     

Photodynamic therapy for the treatment of advanced gastrointestinal tumours

In the study, 120 patients with advanced gastrointestinal tumours were treated by PDT; 5 mg/kg of HpD was intravenously given 48–72 h prior to PDT. The light source was an argon dye laser with an output beam of 630 nm. The irradiation time varied from 15–25 min with a power of 100–350 mW cm^–2. The entire tumour was irradiated with a light dose of 100–250 J cm^–2. Of the 120 patients, 20 had cancer of esophagus, 72 had cancer of the gastric cardia, 18 had cancer of the stomach and 10 had cancer of the rectum. Eighty-eight patients (73.3%) had a response to PDT. Twelve patients with CR were followed up for one to five years, two patients died during the two years after PDT.     

Thermal effects of CO2 laser on the pulpal chamber and enamel of human primary teeth: An in vitro investigation

Background and Objective: Thermal effects of direct exposure of the pulp chamber to CO₂ laser irradiation were evaluated and potentially safe parameters for its clinical application were determined. Study Design/Meterials and Methods: Using conventional methods, the pulp chambers of freshly extracted primary cuspids were exposed and then subjected to CO₂ laser irradiation at the following parameters: 2, 4, 6, 8, 10, 15, and 20 Watts power settings; 0.76 mm diameter spot size; total energy densities between 4–1,058 J/cm²; exposure time between.01–60 s. Exposures in the continuous wave mode were performed in some specimens. In others, single firings of a.01 s pulse duration and a 1 s pulse interval were conducted. Temperature measurements were conducted using a thermocouple and thermal camera. Results: Significant statistical differences in maximum temperature rise on enamel were observed in groups that received total energy densities ≥ 8 J/cm² (P ≤ 0.01); within the pulp chambers these were observed in groups that used ≥ 44 J/cm² (P ≤ 0.01). Conclusion: Parameters appropriate for pulpotomy were observed at energy densities up to 176 J/cm² in the continuous wave mode and 264 J/cm² using single pulses, wherein temperature rises below 5.5°C were recorded on tooth surface. © 1995 Wiley-Liss, Inc.     

Differences between cytotoxicity in photodynamic therapy using a pulsed laser and a continuous wave laser: study of oxygen consumption and photobleaching

Oxygen consumption at the targeted site has a significant effect on dosimetry in photodynamic therapy (PDT). However, oxygen consumption in PDT using a pulsed laser as a light source has not been clarified. We therefore investigated the dependence of cytotoxicity on the oxygen consumption and the photosensitizer photobleaching of PDT using a pulsed laser by comparing with that using a continuous wave (CW) laser. Mouse renal carcinoma cells (Renca) were incubated with a second-generation photosensitizer, PAD-S31. The cells were then irradiated with either a 670-nm nanosecond pulsed light from the 3rd harmonics of a Nd:YAG laser-pumped optical parametric oscillator with a peak fluence rate of ~1 MW/cm² at 30 Hz or a 670-nm CW diode laser with a total light dose of 40 J/cm². Regardless of laser source, cytotoxic effects exhibited cumulative dose responses to the photosensitizer ranging from 12 to 96 g/ml. However, cytotoxic effect of PDT using the pulsed light was significantly less than that using the CW light with the photosensitizer concentrations of 24 and 48 g/ml under identical fluence rates. During PDT, the cells exposed to the pulsed light consumed oxygen more slowly, resulting in a lower amount of oxygen consumption when compared with PDT using CW light. In accordance with oxygen consumption, the pulsed light induced significantly less photobleaching of the photosensitizer than the CW light did. These results indicate that the efficiency of PDT using pulsed light is less when compared with CW light, probably being related to suppressed oxygen consumption during the pulsed light irradiation.