Donna J. Castro

Interstitial laser phototherapy assisted by magnetic resonance imaging: A new technique for monitoring laser-tissue interaction

The rapid technological advances of magnetic resonance imaging, laser fiberoptics, and compatible probes may allow treatment of deep and sometimes surgically unreach‐able tumors of the head and neck with minimal morbidity through interstitial laser phototherapy. In this study, a new application of magnetic resonance imaging was developed to monitor and quantify laser‐induced tissue damages. Pig skin was exposed to increased levels of argon laser (514.5 ran) at energy densitites between 62.5 and 375 J/cm2 as determined by an accurate and reproducible method of dosimetry. Thermal profiles were recorded using an infrared sensor and Tl‐ and T2‐weighted magnetic resonance images were taken; afterward, biopsies were performed to quantitate the level of tissue damage. Our results demonstrate that above a certain threshold of laser energy, the magnetic resonance imaging findings are temperature dependent. Appropriate development of a scale matching laser energies, temperature profiles, Tl‐ and T2‐weighted magnetic resonance images, and histological quantitation of tissue destruction will allow us to optimize the three‐dimensional control and monitoring of laser‐tissue interactions.

Laser dyes for experimental phototherapy of human cancer: Comparison of three rhodamines

The mitochondrial dye Rhodamine 123 (Rh‐123) has been shown to be an effective photosensitizer for argon‐laser irradiation of some types of human cancer cells in vitro. We reported that 514.5‐nm laser illumination of Rh‐123 sensitized human melanoma, and squamous carcinoma cells strongly inhibited tumor‐cell proliferation as measured by decreased 3H‐thymidine (3H‐T) uptake in vitro and may eradicate some tumors when grown as transplants in nude mice. However, several other human tumors were resistant to Rh‐123 laser therapy in vitro and in vivo. In the current study, it was possible to obtain 100‐ to 1000‐fold increased sensitivity to 514.5‐nm laser illumination by replacement of Rh‐123 with the cationic rhodamine dyes Rh‐3G and Rh‐6G. Cell viability was decreased over 95% and 3H‐T incorporation reduced at least 80% by laser phototherapy after sensitizing tumor cells with 1 μg/mL Rh‐123, 0.01 μg/mL Rh‐3G, or 0.001 μg/mL Rh‐6G. However, Rh‐123 alone did not decrease 3H‐T uptake significantly unless present at over 10‐ to 100‐fold higher levels than Rh‐3G, respectively. The tumor cell dye uptake level was measured by N‐butanol extraction and absorption scans at 400 to 600 nm. The results revealed that dye uptake was more rapid, and retention of Rh‐3G and Rh‐6G was 5‐ to 10‐fold higher than for Rh‐123 in the human tumor cells. The data suggest that Rh‐3G and Rh‐6G may be highly sensitive chromophores for laser phototherapy of human cancer cells.

Rhodamine-123 as a new photochemosensitizing agent with the argon laser: nonthermal and thermal effects on human squamous carcinoma cells in vitro

A human squamous carcinoma cell line (P3) was first exposed to a nontoxic dose of Rhodamine‐123 (1 μg/m1 for 1 hour), then subjected to treatment with a single mode argon laser at 514.5 nm. The temperature and energy levels delivered to the target cells were determined by a reproducible method of dosimetry. Cell viability was assessed by the trypan blue exclusion test. Cell duplication and DNA synthesis were measured by the incorporation of 3H‐thymidine at 6 and 24 hours post‐treatment. The results indicate that Rhodamine‐123 at nontoxic doses of 1 μ/m1 enhanced the tumoricidal effects of the argon laser at reduced temperatures as low as 40 °C. Furthermore, at physiological temperature ranges as low as 28 to 39 °C, an immediate and/or delayed inhibition of cell duplication was demonstrated, while cell viability was not affected. These observations, suggest that Rhodamine‐123 can be used effectively as a chemosensitizing agent in the treatment of human tumor cells with the argon laser at 514.5 nm. This new technique of tumor cell targeting by Rhodamine sensitization and specific laser treatment may offer real advantages without the extreme photosensitivity associated with hematoporphyrin derivatives.

Future directions of laser phototherapy for diagnosis and treatment of malignancies: Fantasy, fallacy, or reality?

A new and highly promising adjunctive modality for the diagnosis and therapy of malignancies is under development using lasers and tumor targeting dyes. To reach the eventual goal of clinical treatment, several current “fantasies and fallacies” regarding laser applications in medicine must be identified and their problems clearly outlined. A multidisciplinary scientific approach is also required to enable the clinical practicality of this laser targeting approach. Many new dyes and laser wavelengths are being tested to improve specific tumor uptake and/or retention, lower systemic toxicity, increase tissue penetration, and identify fluorochromes with synergistic properties to further enhance laser tumoricidal effects. Rapid technological advancements in magnetic resonance imaging may now provide an extremely sensitive way to detect and monitor laser‐tissue effects, and allow efficient interstitial laser phototherapy of deep and sometimes inaccessible tumors. The current and future prospectives of the emerging field of laser phototherapy are described.

Biostimulative effects of ND:YAG q-switch dye on normal human fibroblast cultures: Study of a new chemosensitizing agent for the ND:YAG laser†

Kodak Q‐switch II® is a new chemical with an absorption maxima at 1,051 nm, designed to be used as an Nd:YAG dye laser. The potential for this dye as a new chemosensitizing agent in the treatment of connective tissue diseases and wound healing with low energy Nd:YAG laser was examined. Two normal fibroblast cell lines were tested for sensitivity to various levels of this dye in vitro. These cells were exposed to Q‐switch II dye at concentrations of 0.01, 0.1, 1, 10, 50, and 100 μg/ml for 1 and 24 hours. Cell viability was assessed by the trypan blue exclusion test. Cell duplication and DNA synthesis were measured by the incorporation of [3H]‐thymidine at 6 and 24 hours postexposure to Q‐switch II dye. At concentrations up to 10 μg/ml, both cell lines tested showed no changes in cell viability. However, at concentrations equal or higher than 50 μg/ml, more than 40% of the fibroblasts incorporated trypan blue after 24 hours of exposure to this dye, indicating significant cell destruction. The results indicate that Q‐switch II dye is nontoxic to normal human fibroblast cultures and showed significant biostimulative effects on cell duplication at concentrations equal to or lower than 10 μg/ml. Further studies will be required to determine the usefulness of Q‐switch EC dye as a new photochemosensitizing agent for potential biostimulation of wound healing and/or treatment of connective tissue diseases with the Nd:YAG laser (near infrared, 1,060 nm) at “nonthermal” levels of energies.

Biodistribution of rhodamine-123 in nude mice heterotransplanted with human squamous cell carcinomas

Rhodamine‐123 uptake and release was determined in nu/nu mice heterotransplanted with P3 human squamous carcinomas to assess its value as an in vivo laser photosensitizer for treatment of solid tumors. Following intraperitoneal injection of Rh‐123 (1 μg/g of body weight), mice were killed at 2, 4, 6 and 24 hours, and 3 and 7 days postinjection. The peak concentrations of Rh‐123 per milligram of tissue measured by fluorescence spectrophotometry was distributed as follows: kidneys>spleen>intestine> stomach>liver>tumor>skin>skeletal muscles> lung>heart>blood>brain. No preferential uptake or retention of Rh‐123 by tumors was observed. However, a longer retention with higher concentrations of the dye was seen in normal skin as opposed to P3 tumors from 4 hours to 7 days postinjection with Rh‐123. The elimination of Rh‐123 was rapid, with the dye falling to less than 2% of peak concentration at 7 days postinjection. Knowledge of Rh‐123 biodistribution in tumors and other tissues suggests that optimal timing after injection of this dye may allow selective photodiagnosis and photodynamic therapy of tumors with the argon laser.

Phototherapy with the argon laser on human melanoma cells “sensitized” with rhodamine-123: A new method for tumor growth inhibition†

Laser photodynamic therapy of superficial malignancies is a promising new approach that will become clinically useful when fluorochromes with high tumor specificity and low toxicity to normal tissues are identified. We recently reported that the mitochondrial dye, Rhodamine‐123 (Rh‐123), at nontoxic doses, is an effective sensitizing agent for argon laser treatment of human squamous carcinoma and melanoma cells in vitro. We now report the complete inhibition of in vivo tumor development by human M24 melanoma cells transplanted subcutaneously into nu/nu mice after exposure to 1μg/ml of Rh‐123 for 1 hour and treatment with an argon laser at nonthermal temperatures of 38 to 40 °C. Significant in vivo growth was observed for all control tumors. These results demonstrate that Rh‐123 enhances the tumoricidal effects of the argon laser at nonthermal temperatures and provides evidence that effective photodynamic therapy may be possible in vivo with the new fluorochrome Rhodamine‐123.

Bioinhibition of human fibroblast cultures sensitized to Q‐switch II dye and treated with the Nd:YAG laser: A new technique of photodynamic therapy with lasers

Kodak Q‐switch II dye has recently proven to be nn effective biostimulative agent on normal human fibroblast cultures. The potential for this dye as a new chemosensitizing agent for the treatment of connective tissue diseases and wound healing with the Nd:YAG laser was examined. Two normal fibroblast cell lines were first sensitized to a nontoxic dose of Q‐switch II dye, then subjected to treatment with an Nd:YAG laser at 1, 060 nm, with varying levels of energy and temperatures determined by a reproducible method of dosimetry. The results indicate that Q‐switch II dye at nontoxic doses of 0.1 μg/ml enhances the cytotoxic effects of the Nd:YAG laser at temperatures as low as 36 °C. Furthermore, at physiological temperature ranges as low as 24 °C to 34 °C, cell duplication was inhibited, but cell viability was not affected. Similar results were not observed when fibroblast cultures were treated with the laser alone. These observations suggest that Q‐switch II dye is an effective chemosensitizing agent for the Nd:YAG laser and could potentially be used to reduce collagen deposits in conditions such as keloids and hypertrophic scars.

The effects of argon lasers on human melanoma cells sensitized with rhodamine-123 in vitro.

A human melanoma cell line, M14, was first exposed to a nontoxic dose of Rhodamine-123 (1 microgram/ml) for one hour, then subjected to a treatment with a single mode argon laser at 514.5 nm. The temperature and energy levels delivered to the target cells were determined by a reproducible method of dosimetry. Cell viability was assessed by the Trypan Blue exclusion test. Cell duplication and DNA synthesis were measured by the incorporation of 3H-thymidine at 6 and 24 hours post-treatment. At energy levels and temperatures higher or equal to 950 J/cm2 (40 degrees C), an immediate suppression of DNA synthesis was accompanied by nonviability of the M14 carcinoma cells. At energy levels between 130-900J/cm2 corresponding to temperatures between 28 to 39 degrees C, both an immediate and delayed inhibition of DNA synthesis was noted but the cells remained viable. The results indicate that Rhodamine-123 at nontoxic doses of 1 microgram/ml enhances the tumoricidal effects of the argon laser at reduced temperatures as low as 40 degrees C. Furthermore, at physiological temperature ranges as low as 28 to 30 degrees C, an immediate inhibition of cell duplication was demonstrated while cell viability was not affected. These observations suggest that Rhodamine-123 can be used effectively as a chemosensitizing agent in the treatment of human tumor cells with the argon laser at 514.5 nm.

Photodynamic therapy using rhodamine-123 as a new laser dye: Biodistribution, metabolism and histology in new zealand rabbits

Rhodamine-123 (Rh-123) has been tested recently as a new laser dye for photodynamic therapy of human tumors in vitro and in vivo. Prior to initiation of clinical studies of this technique, we evaluated the biodistribution, metabolism, and pathological changes of Rh-123 in rabbits after systemic, repetitive injections of the dye in escalating doses. At doses between 0.1 to 1 mg/kg of Rh-123 injected intramuscularly (IM) daily for 5 days, no local or systemic toxicity was observed during the 4 weeks of follow-up. The peak concentrations of Rh-123 in micrograms/g of tissue was distributed as follows: kidney (3.24) greater than heart (2.24) greater than spleen (1.77) greater than lung (0.61) greater than liver (0.38) greater than skin (0.30) greater than skeletal muscle (0.17) greater than genitals (0.13) greater than brain (0.04). The elimination of Rh-123 was very rapid, with the dye falling to 2.7% of peak concentration at 72 hours in the kidneys, and to undetectable levels at 240 hours postinjection in all organs, except the skin, which retained 3% of the peak level at 240 hours. The low toxicity and rapid metabolism of Rh-123 in this preclinical model suggests that the dye and Argon laser may represent an effective combination for treatment of superficial malignancies.