Fluorescence diagnosis and photodynamic therapy in dermatology from experimental state to clinic standard methods.

The role of photodynamic therapy (PDT) in the treatment of in situ neoplasias and tumors of the skin is steadily increasing. An intratumoral enriched photosensitizer and its activation by light are the principles of photodynamic action. Aminolevulinic acid (ALA) has been shown to be the drug with most experimental and clinical use in the past. The highest efficacy with most selectivity in topical PDT is postulated for methyl aminolevulinate or methyl aminooxopenoat (MAL, MAOP, Metvix). For solar keratoses, topical PDT using MAL is already considered to be the treatment of choice. Epithelial skin tumors such as basal cell carcinomas also respond very well, however, a debulking procedure of the exophytic tumor tissue is an absolute prerequisite to a successful cure. In addition to functioning as a novel therapeutic tool, photodynamic sensitization of skin cancer cells is increasingly used for fluorescence diagnosis (FD) (also known as photodynamic diagnosis or PDD). The fluorescence of induced porphyrins is effective in detecting and delineating neoplastic skin areas. Future approaches of FD and PDT are nontumoral applications, especially psoriasis, viral-induced diseases, or acne vulgaris. Topical PDT is well tolerated and leads to excellent aesthetic results with only minor side effects.     

Fluorescence detection of protoporphyrin IX in living cells: a comparative study on single- and two-photon excitation

Photodynamic therapy (PDT) involves a combination of a lesion-localizing photosensitizer with light and has been established as a new modality for some medical indications. Much evidence has shown the correlation between subcellular localization of a photosensitizer with its photodynamic efficiency. However, the fluorescence of most photosensitizers in cells is weak and easily photobleached. We compare the effect of single-photon excitation (SPE) with that of two-photon excitation (TPE) on fluorescence detection of protoporphyrin IX (PpIX), a potent photosensitizer, in the PLC hepatoma cells in vitro. By using laser scanning confocal fluorescence microscopy, both fluorescence images and spectra of intracellular PpIX are studied with SPE of 405- and 488-nm lasers, and TPE of 800-nm femtosecond laser. The 405-nm laser is more efficient at exciting PpIX fluorescence than the 488-nm laser, but causes a considerable photobleaching of the PpIX fluorescence and induces weak autofluorescence signals of native flavins in the cells as well. The 800-nm TPE is found to significantly improve the quality of PpIX fluorescence images with negligible PpIX photobleaching and minimized endogenous autofluorescence, indicating the potential of 800-nm TPE for studying cellular localization of porphyrin photosensitizers for PDT.     

Spectroscopic measurements of photoinduced processes in human skin after topical application of the hexyl ester of 5-aminolevulinic acid.

Although 5-aminolevulinic acid, ALA, and its derivatives, have been widely studied and applied in clinical photodynamic therapy (PDT), there is still a lack of reliable and non-invasive methods and technologies to evaluate physiological parameters of relevance for the therapy, such as erythema, melanogenesis, and oxygen level. We have investigated the kinetics of these parameters in human skin in vivo during and after PDT with the hexyl ester of ALA, ALA-Hex. Furthermore, the depth of photosensitizer (protoporphyrin IX, PpIX) production after different application times was investigated. It was found that the depth increased with increasing application time of ALA-Hex. We also investigated the depth of PpIX before and after light exposure causing 50% photobleaching at 407 nm. The PpIX localized in superficial layers of the normal tissue was removed during the bleaching. Thus, after bleaching, the remaining PpIX was localized mainly in the deeper layers of normal tissue. We have applied fluorescence emission spectroscopy, fluorescence excitation spectroscopy, and reflectance spectroscopy in the study of the above-mentioned parameters. In conclusion, fluorescence excitation spectroscopy and reflectance spectroscopy are simple, useful, reliable, and noninvasive techniques in the evaluation of the processes taking place in human skin in vivo during and after PDT. Using these methods we were able to quantify melanogenesis, O2 level, erythema, vasoconstriction, and vasodilatation.     

Facilitated delivery of ALA to inaccessible regions via bioadhesive patch systems.

5-Aminolevulinic acid (ALA) is now being increasingly used in photodynamic therapy (PDT) and photodiagnosis (PDD) of lesions of the oral cavity, esophagus, and female genital tract. Clearly, topically applied creams and solutions are not the most suitable drug delivery systems for such moist and irregularly shaped areas, where shear forces may be high. As a result, such areas of the body are currently relatively inaccessible to efficient topical delivery of ALA. The use of a bioadhesive patch for topical delivery of ALA to the lower female genital tract and now, for the first time, to the human lip, is described. Use of the patch, containing 38 mg ALA cm-2, allowed successful PDT of vulval intraepithelial neoplasia lesions and induced protoporphyrin IX production in Herpes simplex lesions of the lip.     

新型卟啉类光敏药物介导的PDT对两种胃癌细胞HGC27和MGC803的杀伤效应

目的 观察本实验室自行合成的新型卟啉类光敏药物对2种人胃癌细胞HGC27和MGC803的光动力学治疗(PDT)作用及作用机制.方法 以人胃癌细胞HGC27和MGC803为实验细胞株.实验分为4组:空白对照组(无药物孵育、无光照),单纯光敏药物组(药物孵育、无光照),单纯光照组(无药物孵育、有光照),光敏药物+光照组(药...     

Correlation of in vivo photosensitizer fluorescence and photodynamic-therapy-induced depth of necrosis in a murine tumor model

We compared light-induced fluorescence (LIF) to nominal injected drug dose for predicting the depth of necrosis response to photodynamic therapy (PDT) in a murine tumor model. Mice were implanted with radiation-induced fibrosarcoma (RIF) and were injected with 0, 5, or 10 mg/kg Photofrin. 630-nm light (30 J/cm(2), 75 mW/cm(2)) was delivered to the tumor after 24 hours. Fluorescence emission (lambda(excitation)=545 nm, lambda( emission)=628 nm) from the tumor was measured. The LIF data had less scatter than injected drug dose, and was found to be at least as good as an injected drug dose for predicting the depth of necrosis after PDT. Our observations provide further evidence that fluorescence spectroscopy can be used to quantify tissue photosensitizer uptake and to predict PDT tissue damage.     

Protoporphyrin IX fluorescence photobleaching increases with the use of fractionated irradiation in the esophagus

Fluorescence measurements have been used to track the dosimetry of photodynamic therapy (PDT) for many years, and this approach can be especially important for treatments with aminolevulinic-acid-induced protoporphyrin IX (ALA-PpIX). PpIX photobleaches rapidly, and the bleaching is known to be oxygen dependent, and at the same time, fractionation or reduced irradiance treatments have been shown to significantly increase efficacy. Thus, in vivo measurement of either the bleaching rate and/or the total bleaching yield could be used to track the deposited dose in tissue and determine the optimal treatment plans. Fluorescence in rat esophagus and human Barrett's esophagus are measured during PDT in both continuous and fractionated light delivery treatment, and the bleaching is quantified. Reducing the optical irradiance from 50 to 25 mWcm did not significantly alter photobleaching in rat esophagus, but fractionation of the light at 1-min on and off intervals did increase photobleaching up to 10% more (p value=0.02) and up to 25% more in the human Barrett's tissue (p value<0.001). While two different tissues and two different dosimetry systems are used, the data support the overall hypothesis that light fractionation in ALA-PpIX PDT esophageal treatments should have a beneficial effect on the total treatment effect.     

光动力疗法的抗肿瘤机制及光敏剂的研究进展

光动力疗法（PDT）是利用光动力效应对疾病进行诊断与治疗的一种非侵袭性技术,已被用于临床头颈部、乳腺、肺、前列腺及皮肤等部位肿瘤的治疗.与传统治疗方法相比,PDT具有创伤小、毒性低、选择性好、适用范围广及不易产生耐药等优势,因而受到肿瘤治疗领域的广泛关注.PDT的抗肿瘤机制复杂,光敏剂是发挥其光动力学效应的关键因素之一,提高光敏剂的靶向输送和携氧能力是改善光动力疗效的重要途径.对PDT的抗肿瘤机制及光敏剂的研究进展进行综述.     

Fluorescence monitoring of a topically applied liposomal Temoporfin formulation and photodynamic therapy of nonpigmented skin malignancies.

Meso-tetra(hydroxyphenyl)chlorin (mTHPC) (INN: Temoporfin) is a potent photodynamically active substance in clinical use today. Usually, the substance is given systemically and a known drawback with this administration route is a prolonged skin light sensitization. For the first time to our knowledge, a liposomal Temoporfin gel formulation for topical application was studied in connection with photodynamic therapy (PDT) of nonpigmented skin malignancies in humans. Intervals of 4 hr between drug administration and light irradiation were used. Sensitizer distribution within tumor and surrounding normal skin was investigated by means of point monitoring and imaging fluorescence spectroscopy before, during, and after PDT, showing high tumor selectivity. Furthermore, the bleaching of Temoporfin was studied during the PDT procedure by monitoring the fluorescence following excitation by using a therapeutic light. A 30-35% light-induced photometabolization was shown. No pain occurred during or after treatment. It was also observed that the treated area did not show any swollen tissue or reddening, as is often seen in PDT using topical delta-aminolevulinic acid. On controlling the patients one week after treatment, healing progress was observed in several patients and no complications were registered.     

Monte Carlo modeling of in vivo protoporphyrin IX fluorescence and singlet oxygen production during photodynamic therapy for patients presenting with superficial basal cell carcinomas

We present protoporphyrin IX (PpIX) fluorescence measurements acquired from patients presenting with superficial basal cell carcinoma during photodynamic therapy (PDT) treatment, facilitating in vivo photobleaching to be monitored. Monte Carlo (MC) simulations, taking into account photobleaching, are performed on a three-dimensional cube grid, which represents the treatment geometry. Consequently, it is possible to determine the spatial and temporal changes to the origin of collected fluorescence and generated singlet oxygen. From our clinical results, an in vivo photobleaching dose constant, β of 5-aminolaevulinic acid-induced PpIX fluorescence is found to be 14 ± 1 J/cm(2). Results from our MC simulations suggest that an increase from our typical administered treatment light dose of 75-150 J/cm(2) could increase the effective PDT treatment initially achieved at a depth of 2.7-3.3 mm in the tumor, respectively. Moreover, this increase reduces the surface PpIX fluorescence from 0.00012 to 0.000003 of the maximum value recorded before treatment. The recommendation of administrating a larger light dose, which advocates an increase in the treatment time after surface PpIX fluorescence has diminished, remains valid for different sets of optical properties and therefore should have a beneficial outcome on the total treatment effect.     

Comparison of noninvasive photodynamic therapy dosimetry methods using a dynamic model of ALA-PDT of human skin

A numerical model of ALA photodynamic therapy of human skin was used to calculate photosensitizer fluorescence and singlet-oxygen luminescence (SOL) observable at the skin surface during treatment. From the emissions, three practical dose metrics were calculated: the fractional fluorescence bleaching metric (FFBM) given by F(0)/F, where F is photosensitizer protoporphyrin IX (PpIX) fluorescence and F(0) is its initial value, the absolute fluorescence bleaching metric (AFBM) given by F(0)-F, and the cumulative SOL (CSOL). These three metrics can be measured during clinical PDT treatment, but their relation to actual singlet-oxygen distribution in the skin is complex and may depend on treatment parameters such as irradiance. Using the model, the three metrics were compared to the average singlet-oxygen dose in the dermis. Despite the complex dependence of (1)O(2) concentration on depth, a roughly linear correlation was found for all three dose metrics. The correlation for the FFBM was not robust when treatment parameters were varied and this metric was especially sensitive to the initial PpIX concentration and its depth dependence. The AFBM was less sensitive to treatment conditions but CSOL demonstrated the best overall performance.     

Photosensitizer-conjugated magnetic nanoparticles for in vivo simultaneous magnetofluorescent imaging and targeting therapy

A major challenge in nanotechnology and nanomedicine is to integrate tumor targeting, imaging, and selective therapy functions into a small single nanoparticle (<50 nm). Herein, photosensitizer-conjugated magnetic nanoparticles with ～20 nm in diameter were strategically designed and prepared for gastric cancer imaging and therapy. The second generation photosensitizer chlorin e6 (Ce6) was covalently anchored on the surface of magnetic nanoparticles with silane coupling agent. We found that the covalently incorporated Ce6 molecules retained their spectroscopic and functional properties for near-infrared (NIR) fluorescence imaging and photodynamic therapy (PDT), and the core magnetic nanoparticles offered the functions of magnetically guided drug delivery and magnetic resonance imaging (MRI). The as-prepared single particle platform is suitable for simultaneous targeting PDT and in vivo dual-mode NIR fluorescence imaging and MRI of nude mice loaded with gastric cancer or other tumors.     

Spectral Doppler optical coherence tomography imaging of localized ischemic stroke in a mouse model

We report the use of spectral Doppler optical coherence tomography imaging (SDOCTI) for quantitative evaluation of dynamic blood circulation before and after a localized ischemic stroke in a mouse model. Rose Bengal photodynamic therapy (PDT) is used as a noninvasive means for inducing localized ischemia in cortical microvasculature of the mouse. Fast, repeated Doppler optical coherence tomography scans across vessels of interest are performed to record flow dynamic information with high temporal resolution. Doppler-angle-independent flow indices are used to quantify vascular conditions before and after the induced ischemia by the photocoagulation of PDT. The higher (or lower) flow resistive indices are associated with higher (or lower) resistance states that are confirmed by laser speckle flow index maps (of laser speckle imaging). Our in vivo experiments shows that SDOCTI can provide complementary quantified flow information that is an alternative to blood volume measurement, and can be used as a means for cortical microvasculature imaging well suited for small animal studies.     

Diagnosis and treatment of bladder tumors by photodynamic therapy]

Experimental results and clinical trials showed that bladder tumours Tis and pT1a can be diagnosed by photodynamic diagnosis and treated by photodynamic therapy. These photochemical methods are based on a selective photosensitizer uptake in the tumour tissue. After subsequent irradiation, these tissues emit a fluorescence (diagnosis) or can be destroyed by chemically induced oxidative compounds (treatment). Hematoporphyrin derivative and its oligomeric part have been the light-sensitizing drugs the most commonly studied. Interest has now shifted to 5-aminolevulinic acid, or its hexylester form, inducing protoporphyrin IX which shows better tumour localising properties and could avoid side effects. While photodynamic treatment is still an experimental modality, photodynamic diagnosis could achieve a more accurate detection of occult dysplasia compared to white light cystoscopy.     

Clinical Application of Photodynamic Therapy

Photodynamic therapy（PDT） is a new medical technology, the study on photodynamic therapy was in full swing in the past two decade. Scientists have made great progress in it. Photosensitizer,oxygen and light source play important role in photodynamic therapy. PDT is a light activated chemotherapy. A photon is adsorbed by a photosensitizer which moves the drug into an excited state. The excited drug can then pass its energy to oxygen to create a chemical radical called “singlet oxygen”. Singlet oxygen attacks cellular structures by oxidation. Such oxidative damage might be oxidation of cell membranes or proteins. When the accumulation of oxidative damage exceeds a threshold level,the cell begins to die. Photodynamic therapy allows selective treatment of localized cancer. PDT involves administration of a photosensitizer to the patients, followed by delivery of light to the cancerous region. The light activates the agent which kills the cancer cells. Without light,the agent is harmless. As a new therapy,photodynamic Therapy has great Advantage in treating cancers. 1. PDT avoids systemic treatment. The treatment occurs only where light is delivered, hence the patient does not undergo go needless systemic treatment when treating localized disease. Side-effects are avoided, from losing hair or suffering nausea to more serious complications. 2. PDT is selective. The photosensitizing agent will selectively accumulate in cancer cells and not in surrounding normal tissues. Hence ,there is selective targeting of the cancer and sparing of surrounding tissues. 3. when surgery is not possible. PDT kills cancer cells but does not damage collagenous tissue structures,and normal cells will repopulate these structures. Hence,if a patient has cancer in a structure that cannot be removed surgicaily（eg. ,the upper bronchi of the lung） ,PDT can still treat the site. 4. PDT is repeatable. Uniike radiation therapy,PDT can be used again and again. Hence,it offers a means of longterm management of cancer even if complete cure is not attainable.     

Positron emission tomography imaging of tumor response after photodynamic therapy.

Positron emission tomography (PET) imaging is a powerful noninvasive tool allowing physiological and biochemical processes to be investigated in vivo at the molecular level. In the clinics, it is currently being used to detect and stage cancer and to assess tumor response following therapy. In cancer research, at the preclinical level, PET in conjunction with a dedicated high-resolution small animal scanner can play an important role in drug development as well as in the evaluation of novel treatment protocols. In this paper, we review the use of PET in assessing tumor response to photodynamic therapy (PDT) and discuss its potential role in the development of novel photosensitizers. This molecular imaging modality is particularly promising for the real-time evaluation of tumor response to therapy both in terms of treatment efficacy and action mechanism.     

Fluorescence and absorption assessment of a lipid mTHPC formulation following topical application in a non-melanotic skin tumor model

Although the benefits of topical sensitizer administration have been confirmed for photodynamic therapy (PDT), ALA-induced protoporphyrin IX is the only sensitizer clinically used with this administration route. Unfortunately, ALA-PDT results in poor treatment response for thicker lesions. Here, selectivity and depth distribution of the highly potent sensitizer meso-tetra(hydroxyphenyl)chlorin (mTHPC), supplied in a novel liposome formulation was investigated following topical administration for 4 and 6 h in a murine skin tumor model. Extraction data indicated an average [+/- standard deviation (SD)] mTHPC concentration within lesions of 6.0(+/-3.1) ngmg tissue with no significant difference (p<0.05) between 4- and 6-h application times and undetectable levels of generalized photosensitivity. Absorption spectroscopy and chemical extraction both indicated a significant selectivity between lesion and normal surrounding skin at 4 and 6 h, whereas the more sensitive fluorescence imaging setup revealed significant selectivity only for the 4-h application time. Absorption data showed a significant correlation with extraction, whereas the results from the fluorescence imaging setup did not correlate with the other methods. Our results indicate that this sensitizer formulation and administration path could be interesting for topical mTHPC-PDT, decreasing the effects of extended skin photosensitivity associated with systemic mTHPC administration.     

基于氨乙酰丙酸(ALA)脂类衍生物的光动力疗法(PDT)

AL A脂类衍生物是目前光动力疗法领域中最活跃的光敏剂前体物 ,它因能够有效地通过外源加入的方式在肿瘤细胞内内源生成进而积聚的原卟啉 (Pp IX)光敏剂而在光动力疗法领域独树一帜。本文将沿着 AL A脂类衍生物的光动力疗法实验过程这一主线而对它的光动力疗法机理及实验研究结果作一综述。主要包括 :细胞对外源 AL A脂类衍生物的摄取及转化为 AL A的生化机制 ;由 AL A生成内源原卟啉 Pp IX的生化机理 ;由 AL A内源生成的光敏剂引起的光致敏过程     

A hematoporphyrin-based delivery system for drug resistance reversal and tumor ablation

Nanotechnology-based drug delivery systems have been intensively investigated, while only a few of them can be used for clinic application. Hematoporphyrin (HP), a major molecule in erythrocyte, has been widely used in photodynamic therapy (PDT). In the present study, polyethylene glycol (PEG) modified hematoporphyrin (HPP)-based nanoparticle system was designed to load doxorubicin (HPPD), in achieving a synergistic effect of chemotherapy and PDT. Herein we presented that HPPD formed narrowly dispersed nanoparticles at 35 ± 2 nm, yielding an enhanced drug release at pH5.8 along with laser radiation. This combined treatment with HPPD and radiation facilitated drug penetration to the nucleus thereby reducing 12-fold decrease in IC₅₀ value and promoting apoptosis in drug-resistant breast cancer cells. Notably, little toxicity was detected with HPP at the cellular level and in animal models. Live animal imaging revealed that HPPD performed ultra high tumor uptake in both mice and marmoset models. Strikingly, intravenous administration of HPPD and radiation on the tumor achieved efficient tumor ablation, without inducing myocardial injury. We report here the development of a biomolecule, HP-based nanoparticle system, which can synergistically yield chemotherapy and PDT.     

Photodynamic therapy of nodular basal cell carcinoma with multifiber contact light delivery.

To overcome the limited treatment depth of superficial photodynamic therapy we investigate interstitial light delivery. In the present work the treatment light was delivered using a system in which three or six clear-cut fibers were placed in direct contact with the tumor area. This placement was thought to represent a step toward general purpose interstitial PDT. Twelve nodular basal cell carcinomas were treated employing delta-aminolevulinic acid and 635 nm laser irradiation. Fluorescence measurements were performed monitoring the buildup and subsequent bleaching of the produced sensitizer protoporphyrin IX. The treatment efficacy, judged at a 28-month follow-up, showed a 100% complete response. Two punch excisions at 7 months converted two partial responses to complete responses. One patient failed to appear at all follow-up sessions. The outcome of the treatments was comparable to superficial photodynamic therapy in terms of histological, clinical, and cosmetic results.