Qian Peng

Lasers in medicine

It is hard to imagine that a narrow, one-way, coherent, moving, amplified beam of light fired by excited atoms is powerful enough to slice through steel. In 1917, Albert Einstein speculated that under certain conditions atoms could absorb light and be stimulated to shed their borrowed energy. Charles Townes coined the term laser (light amplification by stimulated emission of radiation) in 1951. Theodore Maiman investigated the glare of a flash lamp in a rod of synthetic ruby, creating the first human-made laser in 1960. The laser involves exciting atoms and passing them through a medium such as crystal, gas or liquid. As the cascade of photon energy sweeps through the medium, bouncing off mirrors, it is reflected back and forth, and gains energy to produce a high wattage beam of light. Although lasers are today used by a large variety of professions, one of the most meaningful applications of laser technology has been through its use in medicine. Being faster and less invasive with a high precision, lasers have penetrated into most medical disciplines during the last half century including dermatology, ophthalmology, dentistry, otolaryngology, gastroenterology, urology, gynaecology, cardiology, neurosurgery and orthopaedics. In many ways the laser has revolutionized the diagnosis and treatment of a disease. As a surgical tool the laser is capable of three basic functions. When focused on a point it can cauterize deeply as it cuts, reducing the surgical trauma caused by a knife. It can vaporize the surface of a tissue. Or, through optical fibres, it can permit a doctor to see inside the body. Lasers have also become an indispensable tool in biological applications from high-resolution microscopy to subcellular nanosurgery. Indeed, medical lasers are a prime example of how the movement of an idea can truly change the medical world. This review will survey various applications of lasers in medicine including four major categories: types of lasers, laser-tissue interactions, therapeutics and diagnostics.

Photodynamic therapy with 5-aminoolevulinic acid-induced porphyrins and DMSO/EDTA for basal cell carcinoma

Seven hundred sixty three basal cell carcinomas (BCCs) in 122 patients were treated by photodynamic therapy by 5-aminolevulinic acid (ALA) in cream topically applied, either alone, in combination with dimethyl sulphoxide (DMSO) and ethylenediaminetetraacetic acid disodium salt (EDTA), or with DMSO as a pretreatment. After 3 hours cream exposure 40 - 200 Joules/cm2 of 630 nm laser light was given. Fluorescence imaging of biopsies showed highly improved ALA penetration depth and doubled ALA-induced porphyrin production using DMSO/EDTA. Treatment response was recorded after 3 months. After a single treatment 90% of 393 superficial lesions responded completely, independent of using DMSO/EDTA. In 363 nodulo-ulcerative lesions the complete response rate increased from 67% to above 90% with DMSO/EDTA for lesions less than 2 mm thickness and from 34% to about 50% for lesions thicker than 2 mm. Recurrence rate observed during a follow-up period longer than 12 months was 2 - 5%. PDT of superficial thin BCCs with ALA-induced porphyrins and DMSO/EDTA equals surgery and radiotherapy with respect to cure rate and recurrence. Cosmetic results of ALA-based PDT seemed to be better than those after other therapies. In patients with the nevoid BCC syndrome the complete response rate after PDT was far lower.

Photodegradation of sensitizers in mouse skin during PCT

All photosensitizers applied in experimental and clinical photochemotherapy (PCT) of cancer are degraded during light exposure. Under certain conditions this may be a disadvantage since larger light fluences are needed to destroy the malignant tissue. However, photodegradation may also offer an advantage: if the applied dose of sensitizer is so low that most of it is photodegraded before normal tissue is destroyed, but still large enough to sensitize the tumor to destruction, one may achieve a larger tumor to normal tissue therapeutic ratio than when using a higher dose of sensitizer. Tumors usually contain two to ten times higher concentrations of sensitizers than do the surrounding normal tissues. We have studied the photodegradation of a number of sensitizers, including Photofrin (PII), benzoporphyrin derivative mono acid ring A (BPD), chlorin e6 (Chle6) 5-aminolevulinic acid (ALA)- induced protoporphyrin IX (PpIX), meso-tetrahydroxyphenyl-chlorin (m-THPC), meso- tetrahydroxyphenyl-porphyrin (m-THPP) tetraphenylporphine tetrasulfonated (TPPS4), aluminum phthalocyanine disulfonated (AlPcS2), tetrasulfonated (AlPcS4) and zinc phthalocyanine (ZnPc) in liposomes. The sensitizers were injected in Balb/c nude mice and exposed to light from an argon pumped dye laser, tuned to the appropriate therapeutic wavelength at a fluence rate of 100 mW/cm2. The sensitizer fluorescence in the laser- exposed skin was monitored by a fiberoptic probe coupled to a fluorescence spectrometer. The kinetics of the fluorescence decay during PCT were, in all cases, nonexponential but differed from dye to dye. Chle6 and m-THPC were found to be the most photolabile sensitizers. AlPcS4 and AlPcS2 and, to a minor degree, TPPS4 showed a peculiar fluorescence increase during PCT, similar to what we have found earlier for these sensitizers in cells in vitro. The fluorescence increase is indicative of lysosomal localization and perforation of the lysosomes during PCT.

Localizing and photosensitizing mechanism by tetra(3-hydroxyphenyl)porphin in vivo on human malignant melanoma xenografts in athymic nude mice

Observations on time-dependent localization of tetra(3-hydroxyphenyl)porphin (3-THPP) in human malignant melanoma transplanted to athymic nude mice from 1 to 120 h after intraperitoneal (i.p.) 10 mg kg−1 b.w. administration were made by means of fluorescence microscopy. Fluorescence was found on the membrane of the melanoma cells and in the cytoplasm with a peak fluorescence intensity at 24 h post-injection of 3-THPP. The growth of the tumour cells was obviously inhibited at an early stage after PCT. Morphological changes of the tumour at various intervals after treatment by PCT with 3-THPP were also observed. Diffuse degeneration of the tumour cells with swelling of mitochondria and endoplasmic reticulum, heterochromatin aggregation and margination, etc., and subsequently diffuse necrosis with little or no the background of tumorous vascular response were found at an early stage after PCT. On the other hand, it was also observed that the necrosis of the melanoma areas was caused as a consequence of tumorous vascular injury at a later stage after PCT. Thus, two tumoricidal processes caused by PCT with 3-THPP were seen: a direct phototoxic action on tumour cells at an early stage after PCT and an indirect effect secondary to tumorous vascular injury at a later period after PCT.

Biodistribution, pharmacokinetic, and in-vivo fluorescence spectroscopic studies of photosensitizers

Some key data concerning the pharmacokinetics of PCT photosensitizers are reviewed. The following topics are discussed: The binding of photosensitizers to serum proteins, and the significance of LDL binding for tumor localization, the distribution of sensitizers among different tissue compartments and the significance of extracellular proteins and other stromal elements, such as macrophages, low tumor pH, leaky vasculature and poor lymphatic drainage for tumor selectivity of drugs, the retention and excretion of sensitizers, and intracellular pharmacokinetics. Furthermore, the usefulness of fluorescence measurements in the study of sensitizer pharmacokinetics is briefly discussed. A key observation is that 1O2 has a short radius of action. Since practically all PCT sensitizers act via the 1O2 pathway, only targets with significant sensitizer concentrations can be damaged. A given number of 1O2 entities generated in different organelles (mitochondria, lysosomes, plasma membrane, etc.) may lead to widely different effects with respect to cell inactivation. Similarly, sensitizers localizing in different compartments of tissues may have different photosensitizing efficiencies even under conditions of a similar 1O2 yield.

Subcellular photodynamic action sites of sulfonated aluminum phthalocyanines in a human melanoma cell line

By means of scanning and transmission electron microscopy the subcellular target sites of photodynamic therapy (PDT) with derivatives of sulfonated aluminum phthalocyanine (AlPcS1, AlPcS2, AlPcS3, and AlPcS4) were studied in a human melanoma LOX cell line. It was found that PDT with AlPcS1 or AlPcS2 damaged mainly the biomembraneous system of the cells, such as cytoplasmic membrane, mitochondria, endoplasmic reticulum, etc., while AlPcS3- or AlPcS4- induced PDT largely destroyed the lysosomes. However, none of the AlPcSns led to nuclear damage at an early stage after PDT. The subcellular photodynamic targets of the derivatives of AlPcSn are related to the subcellular localization pattern of the dye in the LOX cells.

Cellular responses to photodynamic therapy

The mechanisms of photoinactivation of NHIK 3025 cells in culture sensitized by sulfonated tetraphenyl porphines (TPPSn) are described. Di- and tetrasulfonated species are mainly located in lysosomes. TPPS1 is located diffusely in the extranuclear space, supposedly bound to endoplasmic reticulum as indicated by electron microscopical findings, and to some extent in lysosomes. After PDT TPPSn penetrates the lysosomal membrane. However, after exposure of cells to TPPS4 and light lysosomal enzymes are inactivated before they eventually can be released to the cytosol. In all cases electron microscopical studies show swollen secondary lysosomes after PDT. TPPSn and light induce accumulation of cells in mitosis. This is due to photochemical damage to the unpolymerized form of tubulin.

Dosimetry and light distribution systems for photodynamic therapy at the Norwegian Radium Hospital

Photodynamic therapy has been used for treatment of malignant cutaneous lesions. Photosensitizer has been topically applied, 5-aminolevulinic acid, with light delivered at 630 nm. A study of response rate to varying light doses was performed, it might seem that light doses of 40 - 50 J/cm2 is sufficient for superficial lesions. For nodular lesions higher light doses are needed. Some patients with tumors in the gastrointestinal tract have been treated with systemic administration of 5-aminolevulinic acid and re-treated with Photofrin in case of unsatisfactory results. From the preliminary material available 5-aminolevulinic acid seems less promising as a photosensitizer for internal lesions compared to dermal lesions.

Measurements of sensitizer fluorescence in patients by means of an ordinary fluorescence spectrometer

An ordinary fluorescence spectrometer equipped with a dichroic mirror and fiber optics can be used to measure sensitizer fluorescence in tumors and normal tissues. Our findings with such an instrument showed that after oral administration of 60 mg/kg 5-aminolevulinic acid (ALA) mucosas and rectal adenomas fluoresced stronger than normal skin. Also basal cell carcinomas (BCCs) showed a stronger porphyrin fluorescence than skin after topical application of ALA. The porphyrin fluorescence of BCCs decayed rapidly during standard laser irradiation at 630 nm. The degradation rate of porphyrins in the tumor was approximately constant down to at least 0.3 mm below the tumor surface, in agreement with an optical penetration depth of more than 1 mm.

Effect of desferrioxamine on production of ALA-induced Protoporphyrin IX in normal mouse skin

Topically applied ALA-PDT for human nodular basal cell carcinoma has encountered some problems, one of which is that the concentration of ALA-induced PpIX is in some cases too low to sensitize complete tumor destruction. Our present study, by means of an optical-fiber based point monitoring system in situ, has shown that the fluorescence intensity of PpIX was significantly higher in the normal skin of nude mice after topical application of ALA and desferrioxamine (DF) or in combination with DMSO than that of ALA alone or ALA plus DMSO. These data indicate that DF has an effect on enhancing the production of ALA- induced PpIX in the mouse skin. Thus, there may clinically be advantages of using ALA and DF over ALA alone in PDT of superficial skin lesions.