Vascular and cellular targeting for photodynamic therapy

Photodynamic therapy (PDT) involves the combination of photosensitizers (PS) with light as a treatment, and has been an established medical practice for about 10 years. Current primary applications of PDT are age-related macular degeneration (AMD) and several types of cancer and precancer. Tumor vasculature and parenchyma cells are both potential targets of PDT damage. The preference of vascular versus cellular targeting is highly dependent upon the relative distribution of photosensitizers in each compartment, which is governed by the photosensitizer pharmacokinetic properties and can be effectively manipulated by the photosensitizer drug administration and light illumination interval (drug-light interval) during PDT treatment, or by the modification of photosensitizer molecular structure. PDT using shorter PS-light intervals mainly targets tumor vasculature by confining photosensitizer localization within blood vessels, whereas if the sensitizer has a reasonably long pharmacokinetic lifetime, then PDT at longer PS-light intervals can induce more tumor cellular damage, because the photosensitizer has then distributed into the tumor cellular compartment. This passive targeting mechanism is regulated by the innate photosensitizer physicochemical properties. In addition to the passive targeting approach, active targeting of various tumor endothelial and cellular markers has been studied extensively. The tumor cellular markers that have been explored for active photodynamic targeting are mainly tumor surface markers, including growth factor receptors, low-density lipoprotein (LDL) receptors, transferrin receptors, folic acid receptors, glucose transporters, integrin receptors, and insulin receptors. In addition to tumor surface proteins, nuclear receptors are targeted, as well. A limited number of studies have been performed to actively target tumor endothelial markers (ED-B domain of fibronectin, VEGF receptor-2, and neuropilin-1). Intracellular targeting is a challenge due to the difficulty in achieving sufficient penetration into the target cell, but significant progress has been made in this area. In this review, we summarize current studies of vascular and cellular targeting of PDT after more than 30 years of intensive efforts.     

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.     

Assessment of cellular damage by comet assay after photodynamic therapy in vitro

The aim of this study was analysis of DNA damage in the cell line of the human melanoma G361 after photodynamic therapy (PDT) by comet assay. Photodynamic therapy is based on cytotoxic action of sensitizers (10 microM ZnTPPS4 fixed into 1 mM cyclodextrin hpbetaCD) and light with a suitable wavelength. Single-cell gel electrophoresis (SCGE, comet assay) is a rapid and sensitive method for detecting DNA strand breaks at the level of single cells. Great amount of DNA damage was detected with the dose of irradiation of 0.1; 0.5 J and 2.5 J x cm(-2). Only radiation dose of visible light in the presence of sensitizers can induce DNA breaks of tumour cells. Cells with DNA damage appear as fluorescent comets with tails of DNA fragmentation. In contrast, cells with undamage DNA appear as round spots, because their intact DNA does not migrate out of the cell.     

Immune response against angiosarcoma following lower fluence rate clinical photodynamic therapy.

Tumor response to photodynamic therapy (PDT) is dependent on treatment parameters used. In particular, the light fluence rate may be an important determinant of the treatment outcome. In this clinical case report, we describe the response of angiosarcoma to PDT carried out using different fluence rates and drug and light doses. A patient with recurrent multifocal angiosarcoma of the head and neck was recruited for PDT. A new generation chlorin-based photosensitizer, Fotolon, was administered at a dose of 2.0 to 5.7 mg/kg. The lesions were irradiated with 665 nm laser light for a light dose of 65 to 200 J/cm2 delivered at a fluence rate of 80 or 150 mW/cm2. High dose PDT carried out at a high fluence rate resulted in local control of the disease for up to a year; however, the disease recurred and PDT had to be repeated. PDT of new lesions carried out at a lower fluence rate resulted in tumor eradication. More significantly, it also resulted in spontaneous remission of neighboring and distant untreated lesions. Repeat PDT carried out on a recurrent lesion at a lower fluence rate resulted in eradication of both treated and untreated lesions despite the lower total light dose delivered. Immunohistochemical examination of biopsy samples implies that PDT could have activated a cell-mediated immune response against untreated lesions. Subsequent histopathological examination of the lesion sites showed negative for disease. Our clinical observations show that lower fluence rate PDT results in better outcome and also indicate that the fluence rate, rather than the total light dose, is a more crucial determinant of the treatment outcome. Specifically, lower fluence rate PDT appears to activate the body's immune response against untreated lesions.     

Integrating sphere effect in whole bladder wall photodynamic therapy: I. 532 nm versus 630 nm optical irradiation

The optical absorption, scattering and anisotropy coefficients of piglet bladder, with and without Photofrin, and of diseased human bladder were determined in vitro with a double integrating sphere set-up in the wavelength range 450-800 nm. Monte Carlo simulations were performed in a spherical geometry, representing the bladder, using the optical properties at 532 nm and 630 nm determined in vitro. The calculated fluence rates support the fluence rates that were measured at the bladder wall of a piglet during an in vivo whole bladder wall (WBW) irradiation at 532 nm and 630 nm. Fluence rates calculated and measured in vivo at 630 nm are in agreement with those measured previously in clinical photodynamic therapy (PDT) at 630 nm. WBW-PDT with red light (630 nm) will be technically more advantageous than with green light (532 nm) because of a stronger integrating sphere effect, which reduces the variations of the fluence rate at the bladder wall when the isotropic light source is moved away from the centre of the bladder. Since the optical properties show considerable variations from bladder to bladder, and since as a result the light fluence rate at the bladder wall can vary by a factor of 3 to 4 for the same non-scattered light fluence rate, we conclude that in situ light dosimetry during clinical WBW-PDT is a necessity.     

Biophysical aspects of photodynamic therapy.

Over the last three decades photodynamic therapy (PDT) has been developed to a useful clinical tool, a viable alternative in the treatment of cancer and other diseases. Several disciplines have contributed to this development: chemistry in the development of new photosensitizing agents, biology in the elucidation of cellular processes involved in PDT, pharmacology and physiology in identifying the mechanisms of distribution of photosensitizers in an organism, and, last but not least, physics in the development of better light sources, dosimetric concepts and construction of imaging devices, optical sensors and spectroscopic methods for determining sensitizer concentrations in different tissues. Physics and biophysics have also helped to focus on the role of pH for sensitizer accumulation, dose rate effects, oxygen depletion, temperature, and optical penetration of light of different wavelengths into various types of tissue. These are all important parameters for optimally effective PDT. The present review will give a brief, physically based, overview of PDT and then discuss some of the main biophysical aspects of this therapeutic modality.     

Long-term inhibition of intimal hyperplasia using vascular photodynamic therapy in balloon-injured carotid arteries

Flexible treatments for intimal hyperplasia after angioplasty are still needed. The aim of this study was to demonstrate the long-term effects of vascular photodynamic therapy with talaporfin sodium on intimal hyperplasia following interventional injury. Intimal hyperplasia was induced by balloon distension injury to the carotid artery in 31 rabbits. Talaporfin, 5.0 mg/kg, was delivered systemically immediately after balloon injury. The injury site was irradiated with a diode laser light of wavelength 664 nm using a fluence of 50 J/cm² after 30 min. At day 3 and weeks 3, 6, 9, 15, and 25 after photodynamic therapy, the treated artery of each rabbit was excised and examined immunohistochemically. Thirty minutes after talaporfin administration, drug fluorescence was found only in the balloon-injured carotid artery wall. At 3 days, no smooth muscle cells were seen in the media of the photodynamic therapy-treated arterial segments. Intimal hyperplasia developed progressively in the balloon-injured and untreated segments; however, in the segments treated with photodynamic therapy, intimal hyperplasia was markedly suppressed until 25 weeks and the media was repopulated by smooth muscle cells without macrophages. Vascular photodynamic therapy with talaporfin may be used to inhibit restenosis after vascular intervention. An erratum to this article is available at .     

Immune response after photodynamic therapy increases anti-cancer and anti-bacterial effects

Photodynamic therapy(PDT) is a clinically approved procedure for treatment of cancer and infections. PDT involves systemic or topical administration of a photosensitizer(PS), followed by irradiation of the diseased area with light of a wavelength corresponding to an absorbance band of the PS. In the presence of oxygen, a photochemical reaction is initiated, leading to the generation of reactive oxygen species and cell death. Besides causing direct cytotoxic effects on illuminated tumor cells, PDT is known to cause damage to the tumor vasculature and induce the release of pro-inflammatory molecules. Pre-clinical and clinical studies have demonstrated that PDT is capable of affecting both the innate and adaptive arms of the immune system. Immune stimulatory properties of PDT may increase its beneficial effects giving the therapy wider potential to become more extensively used in clinical practice. Be-sides stimulating tumor-specific cytotoxic T-cells capable to destroy distant untreated tumor cells, PDT leads to development of anti-tumor memory immunity that can potentially prevent the recurrence of cancer. The immunological effects of PDT make the therapy more effective also when used for treatment of bacterial infections, due to an augmented infiltration of neutrophils into the infected regions that seems to potentiate the outcome of the treatment.     

Real-time optoacoustic monitoring of vascular damage during photodynamic therapy treatment of tumor

The optoacoustic technique is a noninvasive imaging method with high spatial resolution. It potentially can be used to monitor anatomical and physiological changes. Photodynamic therapy (PDT)-induced vascular damage is one of the important mechanisms of tumor destruction, and real-time monitoring of vascular changes can have therapeutic significance. A unique optoacoustic system is developed for neovascular imaging during tumor phototherapy. In this system, a single-pulse laser beam is used as the light source for both PDT and for concurrently generating ultrasound signals for optoacoustic imaging. To demonstrate its feasibility, this system is used to observe vascular changes during PDT treatment of chicken chorioallantoic membrane (CAM) tumors. The photosensitizer used in this study is protoporphyrin IX (PpIX) and the laser wavelength is 532 nm. Neovascularization in tumor angiogenesis is visualized by a series of optoacoustic images at different stages of tumor growth. Damage of the vascular structures by PDT is imaged before, during, and after treatment. Rapid, real-time determination of the size of targeted tumor blood vessels is achieved, using the time difference of positive and negative ultrasound peaks during the PDT treatment. The vascular effects of different PDT doses are also studied. The experimental results show that a pulsed laser can be conveniently used to hybridize PDT treatment and optoacoustic imaging and that this integrated system is capable of quantitatively monitoring the structural change of blood vessels during PDT. This method could be potentially used to guide PDT and other phototherapies using vascular changes during treatment to optimize treatment protocols, by choosing appropriate types and doses of photosensitizers and doses of light.     

Laser radiation control in photodynamic therapy of tumors.

This article presents the ideology and basic principles of designing diagnostic and therapeutic medical systems for treating malignant tumors by means of photosensitizers. The designed system is based on optoelectronic devices, which realize selective laser tumor destruction during photodynamic therapy. A distinguishing feature of this system is that laser-beam formation is performed in real time. This enables one to follow changes in the tumor shape during the treatment. As a result of this study, a photodynamic technique for selective phototreatment was developed and tested. Moreover, we detemined light doses for such treatment.     

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.     

Enhanced cutaneous vascular response in AD subjects under donepezil therapy

OBJECTIVE: Abnormal cutaneous vasodilatory responses to the iontophoresis of vasodilators were previously observed in Alzheimer's disease (AD). We sought to replicate these observations and further identify peripheral vascular components of AD pathology. METHODS: Methacholine chloride (MCh), acetylcholine chloride (ACh), and sodium nitroprusside (SNP) were applied iontophoretically to forearm skin. Laser Doppler imaging of treated areas yielded total perfusion response values. RESULTS: Response to MCh was enhanced 78% ( P=0.003 ) in AD subjects under therapy with the acetylcholinesterase inhibitor (AChEI) donepezil ( N=9 ), relative to age- and sex-matched controls ( N=12 ). Significant increases in perfusion were also observed after application of ACh (68%, P=0.03 ) and SNP (46%, P=0.04 ). CONCLUSIONS: A previous study reported attenuated response to ACh in AD. Paradoxically, we observed a substantially enhanced response that is likely a consequence of donepezil therapy. The increased response to the endothelium-independent vasodilator SNP indicates improved general vasodilatory response, perhaps due to preservation of endogenous ACh by donepezil. Cerebral perfusion in response to functional activation may be improved in this way, suggesting a secondary therapeutic mode of donepezil.     

罗丹明123介导的光动力学疗法预防急性移植物抗宿主病的实验研究

目的：探讨Rh123介导的光动力学疗法(PDT)预防异基因造血干细胞移植急性移植物抗宿主病(aGVHD)的可行性及安全性。 方法： 以C57B/6小鼠为供鼠，BALB/c小鼠为受鼠，建立小鼠异基因骨髓移植的aGVHD模型；混合脾脏淋巴细胞培养(MLC)加Rh123孵育，接受氩离子激光30 mW/cm2照射3 min，再与供者骨髓混合移植给受鼠，观察受鼠移植后造血重建、aGVHD发生情况及病理改变、生存率；流式细胞仪检测MLC细胞CD3+CD69+阳性率。 结果： 光动力学治疗组的aGVHD发生减少，肝、皮肤、肠道病理程度减轻，生存率显著高于未经光动力学治疗组；光动力学处理后的混合淋巴细胞培养24 h后，CD34+CD69+表达明显下降。 结论： Rh123介导的光动力学疗法可有效预防小鼠异基因骨髓移植的aGVHD。     

Redox ratio of mitochondria as an indicator for the response of photodynamic therapy

The effect of photodynamic therapy (PDT) treatment on the metabolic state of tumor mitochondria is investigated by imaging of tumor redox status. PDT is performed using the photosensitizer pyropheophorbide-2-deoxyglucosamide (Pyro-2DG), which utilizes the glucose import pathway. It is found that Pyro-2DG-induced PDT resulting in a highly oxidized state of tumor mitochondria. This is determined from the redox ratio changes derived from the intrinsic oxidized flavoprotein (Fp) and reduced pyridine nucleotide (PN) [i.e., reduced nicotinamide adenine dinucleotide (NADH)] fluorescence signals observed using a cryoimager. Thus, the redox ratio is a sensitive indicator for providing reliable and informative measurements of PDT-induced tissue damage. In the PDT treated region of the tumor, highly oxidized flavoprotein and diminishing NADH fluorescence is detected, suggesting that flavoprotein and NADH are oxidized by singlet oxygen produced in the photosensitization process.     

Effect of hypericin-mediated photodynamic therapy on the expression of vascular endothelial growth factor in human nasopharyngeal carcinoma

Photodynamic therapy (PDT) is currently being used as an alternative treatment modality for various types of cancers. PDT involves the selective uptake and retention of a photosensitizer in the tumor followed by light irradiation of an appropriate wavelength to cause the destruction of tumor cells by the formation of cytotoxic reactive oxygen species. The photosensitizer, hypericin, has shown great potential due to its light-dependent tumor destructive properties. However, as hypericin-mediated PDT primarily targets tumor vasculature, it induces certain pro-angiogenic factors such as vascular endothelial growth factor (VEGF) in the tumor tissue as a result of hypoxia. This study examines the role of hypericin-mediated photodynamic therapy in stimulating the expression of key angiogenesis growth factor VEGF in order to elucidate the process of tumor angiogenesis in nasopharyngeal carcinoma xenografts. We also investigated the effect of angiogenesis inhibitor celebrex on human VEGF levels when combined with hypericin-PDT. These studies were conducted on an in vivo human nasopharyngeal xenograft model. VEGF was measured in the control and hypericin-PDT treated tumors. VEGF levels were found to be higher when the tumors were treated at a 1-h drug-light interval compared to a 6-h interval, due to extensive vascular damage. At 72 h post hypericin-PDT, VEGF levels were upregulated indicating the initiation of regrowth in tumors. The use of angiogenesis inhibitor, celebrex, along with hypericin-PDT downregulated the human VEGF levels suggesting that angiogenesis inhibitors can be used to improve the outcome of hypericin-PDT in nasopharyngeal carcinomas.     

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.