基于氨乙酸丙酸（ALA）脂类衍生物的光动力疗法（PDT）

ALA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（PpIX)光敏剂在光动力疗法领域独 树一帜。本文将沿着ALA脂类衍生物的光动力疗法实验过程这一主线而对这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ALA脂类衍生物的提取及转化为ALA的生化机制；由ALA生成内源原卟啉PpIX的生化机理；由ALA内源生成的光敏剂引起的光致敏过程。     

亚氨基二乙酸修饰四苯基卟啉光敏剂介导的光动力疗法对H22肝癌小鼠体内抗肿瘤作用的研究

目的 评价本实验室自行合成的亚氨基二乙酸修饰四苯基卟啉光敏剂的理化性质及对H22小鼠肝癌的光动力治疗作用.方法 采用紫外分光光度法测定亚氨基二乙酸修饰四苯基卟啉光敏剂的紫外吸收光谱、脂水分配系数和单线态氧产率;以肝癌H22小鼠为研究对象,随机分为空白对照组、光照组、光敏剂组、光动力治疗1次组和光动力治疗2次组,考察亚氨基二乙酸修饰四苯基卟啉光敏剂介导的光动力疗法(PDT)对实体瘤的抑制率和荷瘤动物免疫机能的影响.结果 亚氨基二乙酸修饰四苯基卟啉光敏剂在650 nm波长处存在紫外吸收,脂水分配系数和单线态氧产率分别为2.30和0.52;单纯激光照射和单纯给予亚氨基二乙酸修饰四苯基卟啉光敏剂对肝癌H22小鼠无治疗作用(P＞0.05),而亚氨基二乙酸修饰四苯基卟啉光敏剂介导的光动力疗法对小鼠肝癌H22的生长具有极其显著的抑制作用(P＜0.001),光动力治疗2次组肿瘤抑制率高达95.15％.各治疗组对荷瘤小鼠的体质量增长、肝、脾、肺、胸腺指数几乎无影响.结论 亚氨基二乙酸修饰四苯基卟啉光敏剂单线态氧产率高、两亲性好,介导的PDT抗肿瘤活性强,适合作为光动力抗肿瘤候选新药进行开发.     

BPD-MA光动力作用诱导兔血管平滑肌细胞凋亡的初步研究

目的：探讨新型光敏剂苯并卟啉衍生物单环酸A（BPD-MA）光动力作用诱导血管平滑肌细胞凋亡及机制。方法：用体外培养的兔血管平滑肌细胞，加浓度0．25mg／ml的光敏剂BPD-MA，经能量密度4．8J／cm^2波长650nm半导体激光照射。免疫组化染色鉴定平滑肌细胞，HE染色观察细胞形态，MTT法测定细胞增殖活性，TUNEL法观察细胞凋亡。结果：在4．8J／cm^2激光能量密度照射下，与空白对照组比较，光动力作用对平滑肌细胞增殖活性有显著地抑制作用（P〈0．01），单纯激光组照射埘平滑肌细胞的增殖则无明显抑制作用（P〉0．05）。BPD-MA光动力作用使光动力组的大多数细胞出现凋亡。结论：凋亡可能是苯并卟啉衍生物单环酸A光动力抑制兔血管平滑肌细胞增殖的关键因素。     

5-ALA诱导白念珠菌生成PpIX与ALA浓度相关性研究

目的 对不同浓度光敏剂5-氨基酮戊酸(5-ALA)在白念珠菌体内生成原卟啉IX (PpIX)进行荧光强度测定,探求PpIX荧光强度与ALA浓度的关系.为光动力治疗白念珠菌选择最佳的光敏剂浓度提供理论依据.方法 实验分为实验组和对照组,将各组白念珠菌悬液与不同浓度5-ALA混合后避光60min孵育.利用共聚焦激光扫描显微镜测量PpIX荧光强度.结果 实验组白念珠菌与不同浓度ALA避光孵育后均有荧光物质PpIX产生,200mg/mL以上ALA浓度组产生的PpIX强度无统计学意义.300mg/mL以下ALA浓度与PpIX生成强度有相关性.对照组没有检测到PpIX.结论 ALA浓度与生成的PpIX荧光强度密切相关.这为临床ALA-PDT治疗白念珠菌疾病提供了实验依据.     

口腔光动力疗法研究进展

光动力学疗法(PDT)是20世纪70年代末形成的治疗新技术,近年来已从实验研究进入到临床应用阶段,其机理是建立在某些特定类型的细胞或生物体能选择性吸收光敏型的药物,在接受特定波长光照射后产生光敏效应的一种治疗方法.目前主要应用于体表和空腔脏器表面的浅表肿瘤的非手术治疗,由于发现光敏剂对肿瘤以外的多种病变组织和病原微生物具有选择件聚集以及新型光敏剂的开发,使光动力疗法在现代临床中应用领域不断扩大.从光动力治疗的效应机制、光动力治疗的光源与光敏剂以及在口腔医学领域中对头颈部肿瘤、黏膜病、口腔致病微生物感染的治疗等方面的应用研究进展进行了综述.     

口腔光动力疗法研究进展

光动力学疗法(PDT)是20世纪70年代末形成的治疗新技术,近年来已从实验研究进入到临床应用阶段,其机理是建立在某些特定类型的细胞或生物体能选择性吸收光敏型的药物,在接受特定波长光照射后产生光敏效应的一种治疗方法.目前主要应用于体表和空腔脏器表面的浅表肿瘤的非手术治疗,由于发现光敏剂对肿瘤以外的多种病变组织和病原微生物具有选择件聚集以及新型光敏剂的开发,使光动力疗法在现代临床中应用领域不断扩大.从光动力治疗的效应机制、光动力治疗的光源与光敏剂以及在口腔医学领域中对头颈部肿瘤、黏膜病、口腔致病微生物感染的治疗等方面的应用研究进展进行了综述.     

口腔光动力疗法研究进展

光动力学疗法(PDT)是20世纪70年代末形成的治疗新技术,近年来已从实验研究进入到临床应用阶段,其机理是建立在某些特定类型的细胞或生物体能选择性吸收光敏型的药物,在接受特定波长光照射后产生光敏效应的一种治疗方法.目前主要应用于体表和空腔脏器表面的浅表肿瘤的非手术治疗,由于发现光敏剂对肿瘤以外的多种病变组织和病原微生物具有选择件聚集以及新型光敏剂的开发,使光动力疗法在现代临床中应用领域不断扩大.从光动力治疗的效应机制、光动力治疗的光源与光敏剂以及在口腔医学领域中对头颈部肿瘤、黏膜病、口腔致病微生物感染的治疗等方面的应用研究进展进行了综述.     

口腔光动力疗法研究进展

光动力学疗法(PDT)是20世纪70年代末形成的治疗新技术,近年来已从实验研究进入到临床应用阶段,其机理是建立在某些特定类型的细胞或生物体能选择性吸收光敏型的药物,在接受特定波长光照射后产生光敏效应的一种治疗方法.目前主要应用于体表和空腔脏器表面的浅表肿瘤的非手术治疗,由于发现光敏剂对肿瘤以外的多种病变组织和病原微生物具有选择件聚集以及新型光敏剂的开发,使光动力疗法在现代临床中应用领域不断扩大.从光动力治疗的效应机制、光动力治疗的光源与光敏剂以及在口腔医学领域中对头颈部肿瘤、黏膜病、口腔致病微生物感染的治疗等方面的应用研究进展进行了综述.     

口腔光动力疗法研究进展

光动力学疗法(PDT)是20世纪70年代末形成的治疗新技术,近年来已从实验研究进入到临床应用阶段,其机理是建立在某些特定类型的细胞或生物体能选择性吸收光敏型的药物,在接受特定波长光照射后产生光敏效应的一种治疗方法.目前主要应用于体表和空腔脏器表面的浅表肿瘤的非手术治疗,由于发现光敏剂对肿瘤以外的多种病变组织和病原微生物具有选择件聚集以及新型光敏剂的开发,使光动力疗法在现代临床中应用领域不断扩大.从光动力治疗的效应机制、光动力治疗的光源与光敏剂以及在口腔医学领域中对头颈部肿瘤、黏膜病、口腔致病微生物感染的治疗等方面的应用研究进展进行了综述.     

口腔光动力疗法研究进展

光动力学疗法(PDT)是20世纪70年代末形成的治疗新技术,近年来已从实验研究进入到临床应用阶段,其机理是建立在某些特定类型的细胞或生物体能选择性吸收光敏型的药物,在接受特定波长光照射后产生光敏效应的一种治疗方法.目前主要应用于体表和空腔脏器表面的浅表肿瘤的非手术治疗,由于发现光敏剂对肿瘤以外的多种病变组织和病原微生物具有选择件聚集以及新型光敏剂的开发,使光动力疗法在现代临床中应用领域不断扩大.从光动力治疗的效应机制、光动力治疗的光源与光敏剂以及在口腔医学领域中对头颈部肿瘤、黏膜病、口腔致病微生物感染的治疗等方面的应用研究进展进行了综述.     

Induction of apoptosis by hexaminolevulinate-mediated photodynamic therapy in human colon carcinoma cell line 320DM.

Photodynamic therapy (PDT) typically involves systemic or topical administration of a tumor-localizing photosensitizer or prodrug and its subsequent activation by visible light. This results primarily in singlet oxygen-induced photodamage to the tumor. 5-Aminolevulinic acid (ALA) and its derivatives have recently been widely used for PDT due to their selective induction in tumor of endogenous protoporphyrin IX (PpIX), a potent photosensitizer. Although ALA-PDT has achieved successful results in the treatment of several clinical oncological and nononcological diseases, the mechanisms of this modality are still not fully elucidated. In the present study, the human colon carcinoma cell line 320DM was treated in vitro with PDT using hexaminolevulinate (HAL), a hexylester of ALA known to be 50 to 100 times more efficient at producing PpIX formation than ALA itself. PpIX production increased with increasing HAL concentrations in the cells and phototoxicity of the cells was enhanced with increasing light (450 nm) doses. HAL-PDT induced apoptotic cell death, as measured by nuclear staining of Hoechst 33342 for fluorescence microscopy, DNA electrophoresis and TdT staining for flow cytometry. PDT with 5 muM of HAL and a light dose of 640 mJ/cm2 produced a 75% apoptotic cell population 40 hr after the treatment. Furthermore, the loss of mitochondrial membrane potential coincident with the release of cytochrome c from the mitochondria into the cytosol led to a rapid activation of caspase-9 and caspase-3 (an executioner), indicating that the selective damage to the mitochondria by HAL-PDT can induce a cytochrome-c-mediated apoptotic response in the 320DM cells.     

New derivatives of 5-aminolevulinic acid for photodynamic therapy: chemical synthesis and porphyrin production in in vitro and in vivo biological systems.

Photodynamic therapy (PDT) has become a new treatment for several oncological and nononcological disorders. This procedure involves systemic or topical administration of a lesion-localizing photosensitizer or prodrug, followed by irradiation with visible light to cause singlet oxygen-induced damage to the target tissue. 5-aminolevulinic acid (ALA) is an endogenous precursor for several photosensitizing porphyrins formed by heme biosynthesis, and has been studied for PDT with promising results for some superficial diseases of the skin and hollow internal organs. Hydrophilic ALA has a limited ability to penetrate certain biological barriers and has a relatively low selectivity for lesions. In addition, its ability to induce intracellular porphyrins has been shown to be low compared to most esters of ALA. This stimulated a search for lipophilic derivatives of ALA to overcome the shortcomings of ALA. Thirty-two new esters of ALA were prepared and their ability to induce porphyrin formation was assessed in the WiDr human carcinoma cell line in vitro and in the normal skin of Balb/c nude mice in vivo. Branched-chain alkyl esters and substituted benzyl esters were found to be the most efficient porphyrin precursors of the compounds studied.     

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.     

Noninvasive fluorescence monitoring of protoporphyrin IX production and clinical outcomes in actinic keratoses following short-contact application of 5-aminolevulinate

Topical 5-aminolevulinic acid (ALA) is widely used in photodynamic therapy (PDT) of actinic keratoses (AK), a type of premalignant skin lesion. However, the optimal time between ALA application and exposure to light has not been carefully investigated. Our objective is to study the kinetics of protoporphyrin IX (PpIX) accumulation in AK after short contact ALA and relate this to erythemal responses. Using a noninvasive dosimeter, PpIX fluorescence measurements (5 replicates) were taken at 20-min intervals for 2 h following ALA application, in 63 AK in 20 patients. Data were analyzed for maximal fluorescent signal obtained, kinetic slope, and changes in erythema. Our results show that PpIX accumulation was linear over time, becoming statistically higher than background in 48% of all lesions by 20 min, 92% of lesions by 1 h, and 100% of lesions by 2 h. PpIX accumulation was roughly correlated with changes in lesional erythema post-PDT. We conclude that significant amounts of PpIX are produced in all AK lesions by 2 h. The linear kinetics of accumulation suggest that shorter ALA application times may be efficacious in many patients. Noninvasive fluorescence monitoring of PpIX may be useful to delineate areas of high PpIX accumulation within precancerous areas of the skin.     

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.     

Effect of (R)L-sulforaphane on 5-aminolevulinic acid-mediated photodynamic therapy

Topical photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA), or so-called ALA-PDT, is a standard procedure in the clinical practice. For optimal treatment of nonmelanoma skin cancer, actinic keratoses and other dermatoses improvements are required because of adverse side effects, which include pruritus, erythema, edema, and pain. (R)L-sulforaphane (SF) is a compound that protects against erythema, but it can also induce DNA fragmentation that leads to cell death by apoptosis. The aim of our study was to investigate whether SF has any impact on protoporphyrin IX (PpIX) production and on PDT effectiveness. We have investigated some relevant properties of SF: its photostability in dimethyl sulfoxide (DMSO), its effect on ALA-induced production of PpIX in A431 human squamous carcinoma cells and in human skin, its effect on the photoinactivation of PpIX sensitized cells, and its effect on the rate of photobleaching of PpIX. SF had no influence on PpIX photodegradation, neither in solution nor in A431 cells. The synthesis of PpIX was increased by SF in human skin, but not in A431 cells. The average increase in PpIX fluorescence in human skin was 18% +/- 6% and 43% +/- 10% for ALA combined with 80 nmol/L SF and 120 nmol/L SF, respectively. Pretreatment with (R)L-sulforaphane before topical ALA-PDT may improve penetration of ALA through the stratum corneum, and, subsequently, increase PpIX synthesis.     

Innovative drug delivery strategies for topical photodynamic therapy using porphyrin precursors.

Photodynamic therapy (PDT) has been extensively investigated as a treatment for tumors and neoplasias of the skin, bladder, mouth, and female reproductive tract. The most convenient drug delivery route, when focusing on the photodynamic treatment of such tumors and neoplasia, is the transdermal. However, with the inherent "barrier function" of the stratum corneum of the skin, drugs with high molecular weight (> 500 Daltons) or extremes of polarity will find it difficult to successfully cross the skin. Therefore, preformed photosensitizers, which are generally large, highly conjugated molecules, are not commonly used in topical PDT. This has led to the idea of endogenous photosensitization using the small (167.8 Daltons), although hydrophilic, 5-aminolevulinic acid (ALA) being the most frequently employed agent in modern topical PDT. Although clinical application of ALA and its bimolecular effects within target cells remain as primary research themes, the design and evaluation of delivery systems required for effective photosensitizer administration have been less well addressed. This paper briefly reviews traditional approaches to topical delivery of ALA and its esters, and highlights several innovative strategies recently employed to increase the efficacy of ALA-PDT.     

Fluorescence photobleaching of ALA and ALA-heptyl ester induced protoporphyrin IX during photodynamic therapy of normal hairless mouse skin: a comparison of two light sources and different illumination schemes

This study investigated photobleaching of protoporphyrin IX (PpIX) induced by 5-aminolevulinic acid (ALA) and ALA-heptyl ester during superficial photodynamic therapy (PDT) in normal skin of the female BALB/c-nu/nu athymic mouse. We examined the effects of two light sources (laser and broadband lamp) and two different illumination schemes (fractionated light and continuous irradiation) on the kinetics of photobleaching. Our results show that light exposure (0-30 minutes, 10 mW/cm2) of wavelengths of approximately 420 nm (blue light) and 635 nm (red light) induced time-dependent PpIX photobleaching for mouse skin of 2% ALA and ALA-heptyl ester. Blue light (10 mW/cm2) caused more rapid PpIX photobleaching than did red light (100 mW/cm2), which is attributed to stronger absorption at 407 nm than at 632 nm for PpIX. In the case of light fractionation, fractionated light induced faster photobleaching compared with continuous light exposure after topical application of 2% ALA and ALA-heptyl ester in vivo. These have been suggested to allow reoxygenation of the irradiated tissue, with a consequent enhancement of singlet oxygen production in the second and subsequent fractions.     

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.     

Repetitive photodynamic therapy of malignant brain tumors.

The probability of achieving local control with current single-shot, intraoperative photodynamic therapy (PDT) treatments of intracerebral gliomas seems improbable due to the length of time required to deliver adequate light fluences to depths of 1-2 cm in the resection margin. Additionally, due to the short doubling time of many malignant gliomas, the kill rate per cell doubling indicates that it seems unlikely that a single treatment would be sufficient to prevent tumor recurrence. Multiple repetitive treatments would therefore seem required. In this publication we primarily review our work examining the effects of repetitive PDT on malignant brain tumor cells both in vitro and in vivo. The in vitro therapy response of human and rat glioma spheroids to 5-aminolevulinic acid (ALA)-mediated PDT in repetitive form was investigated. The results indicated that PDT repeated at relatively long intervals (weeks) was more effective at inhibiting spheroid growth than either daily fractionated PDT or single-treatment regimes. The in vivo response to repetitive treatment was evaluated in a rodent glioma model where BT4C cell line tumors were established in the brains of inbred BD-IX rats. Microfluorometry of frozen tissue sections showed that PpIX is produced with a 10-20:1 tumor to normal tissue selectivity ratio 4 hr after ALA injection. Preliminary evidence of increased efficacy of repetitive PDT and low fluence rate treatment is presented.