Michael R. Hamblin

Photodynamic therapy and anti-tumour immunity

Photodynamic therapy (PDT) uses non-toxic photosensitizers and harmless visible light in combination with oxygen to produce cytotoxic reactive oxygen species that kill malignant cells by apoptosis and/or necrosis, shut down the tumour microvasculature and stimulate the host immune system. In contrast to surgery, radiotherapy and chemotherapy that are mostly immunosuppressive, PDT causes acute inflammation, expression of heat-shock proteins, invasion and infiltration of the tumour by leukocytes, and might increase the presentation of tumour-derived antigens to T cells.

Photodynamic therapy: a new antimicrobial approach to infectious disease?

Photodynamic therapy (PDT) employs a non-toxic dye, termed a photosensitizer (PS), and low intensity visible light which, in the presence of oxygen, combine to produce cytotoxic species. PDT has the advantage of dual selectivity, in that the PS can be targeted to its destination cell or tissue and, in addition, the illumination can be spatially directed to the lesion. PDT has previously been used to kill pathogenic microorganisms in vitro, but its use to treat infections in animal models or patients has not, as yet, been much developed. It is known that Gram-(-) bacteria are resistant to PDT with many commonly used PS that will readily lead to phototoxicity in Gram-(+) species, and that PS bearing a cationic charge or the use of agents that increase the permeability of the outer membrane will increase the efficacy of killing Gram-(-) organisms. All the available evidence suggests that multi-antibiotic resistant strains are as easily killed by PDT as naive strains, and that bacteria will not readily develop resistance to PDT. Treatment of localized infections with PDT requires selectivity of the PS for microbes over host cells, delivery of the PS into the infected area and the ability to effectively illuminate the lesion. Recently, there have been reports of PDT used to treat infections in selected animal models and some clinical trials: mainly for viral lesions, but also for acne, gastric infection by Helicobacter pylori and brain abcesses. Possible future clinical applications include infections in wounds and burns, rapidly spreading and intractable soft-tissue infections and abscesses, infections in body cavities such as the mouth, ear, nasal sinus, bladder and stomach, and surface infections of the cornea and skin.

Mechanisms in photodynamic therapy: part one—photosensitizers, photochemistry and cellular localization

The use of non-toxic dyes or photosensitizers (PS) in combination with harmless visible light that is known as photodynamic therapy (PDT) has been known for over a hundred years, but is only now becoming widely used. Originally developed as a tumor therapy, some of its most successful applications are for non-malignant disease. In a series of three reviews we will discuss the mechanisms that operate in the field of PDT. Part one discusses the recent explosion in discovery and chemical synthesis of new PS. Some guidelines on how to choose an ideal PS for a particular application are presented. The photochemistry and photophysics of PS and the two pathways known as Type I (radicals and reactive oxygen species) and Type II (singlet oxygen) photochemical processes are discussed. To carry out PDT effectively in vivo, it is necessary to ensure sufficient light reaches all the diseased tissue. This involves understanding how light travels within various tissues and the relative effects of absorption and scattering. The fact that most of the PS are also fluorescent allows various optical imaging and monitoring strategies to be combined with PDT. The most important factor governing the outcome of PDT is how the PS interacts with cells in the target tissue or tumor, and the key aspect of this interaction is the subcellular localization of the PS. Examples of PS that localize in mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus and plasma membranes are given. Finally the use of 5-aminolevulinic acid as a natural precursor of the heme biosynthetic pathway, stimulates accumulation of the PS protoporphyrin IX is described.

Biphasic Dose Response in Low Level Light Therapy

The use of low levels of visible or near infrared light for reducing pain, inflammation and edema, promoting healing of wounds, deeper tissues and nerves, and preventing cell death and tissue damage has been known for over forty years since the invention of lasers. Despite many reports of positive findings from experiments conducted in vitro, in animal models and in randomized controlled clinical trials, LLLT remains controversial in mainstream medicine. The biochemical mechanisms underlying the positive effects are incompletely understood, and the complexity of rationally choosing amongst a large number of illumination parameters such as wavelength, fluence, power density, pulse structure and treatment timing has led to the publication of a number of negative studies as well as many positive ones. A biphasic dose response has been frequently observed where low levels of light have a much better effect on stimulating and repairing tissues than higher levels of light. The so-called Arndt-Schulz curve is frequently used to describe this biphasic dose response. This review will cover the molecular and cellular mechanisms in LLLT, and describe some of our recent results in vitro and in vivo that provide scientific explanations for this biphasic dose response.

The Nuts and Bolts of Low-level Laser (Light) Therapy

Soon after the discovery of lasers in the 1960s it was realized that laser therapy had the potential to improve wound healing and reduce pain, inflammation and swelling. In recent years the field sometimes known as photobiomodulation has broadened to include light-emitting diodes and other light sources, and the range of wavelengths used now includes many in the red and near infrared. The term “low level laser therapy” or LLLT has become widely recognized and implies the existence of the biphasic dose response or the Arndt-Schulz curve. This review will cover the mechanisms of action of LLLT at a cellular and at a tissular level and will summarize the various light sources and principles of dosimetry that are employed in clinical practice. The range of diseases, injuries, and conditions that can be benefited by LLLT will be summarized with an emphasis on those that have reported randomized controlled clinical trials. Serious life-threatening diseases such as stroke, heart attack, spinal cord injury, and traumatic brain injury may soon be amenable to LLLT therapy.

Photodynamic therapy for localized infections—-State of the art

Photodynamic therapy (PDT) was discovered over 100 years ago by observing the killing of microorganisms when harmless dyes and visible light were combined in vitro. Since then it has primarily been developed as a treatment for cancer, ophthalmologic disorders and in dermatology. However, in recent years interest in the antimicrobial effects of PDT has revived and it has been proposed as a therapy for a large variety of localized infections. This revival of interest has largely been driven by the inexorable increase in drug resistance among many classes of pathogen. Advantages of PDT include equal killing effectiveness regardless of antibiotic resistance, and a lack of induction of PDT resistance. Disadvantages include the cessation of the antimicrobial effect when the light is turned off, and less than perfect selectivity for microbial cells over host tissue. This review will cover the use of PDT to kill or inactivate pathogens in ex vivo tissues and in biological materials such as blood. PDT has been successfully used to kill pathogens and even to save life in several animal models of localized infections such as surface wounds, burns, oral sites, abscesses and the middle ear. A large number of clinical studies of PDT for viral papillomatosis lesions and for acne refer to its antimicrobial effect, but it is unclear how important this microbial killing is to the overall therapeutic outcome. PDT for periodontitis is a rapidly growing clinical application and other dental applications are under investigation. PDT is being clinically studied for other dermatological infections such as leishmaniasis and mycobacteria. Antimicrobial PDT will become more important in the future as antibiotic resistance is only expected to continue to increase.

Mechanisms in photodynamic therapy: part two-cellular signaling, cell metabolism and modes of cell death.

Photodynamic therapy (PDT) has been known for over a hundred years, but is only now becoming widely used. Originally developed as a tumor therapy, some of its most successful applications are for non-malignant disease. In the second of a series of three reviews, we will discuss the mechanisms that operate in PDT on a cellular level. In Part I [Castano AP, Demidova TN, Hamblin MR. Mechanism in photodynamic therapy: part one-photosensitizers, photochemistry and cellular localization. Photodiagn Photodyn Ther 2004;1:279-93] it was shown that one of the most important factors governing the outcome of PDT, is how the photosensitizer (PS) interacts with cells in the target tissue or tumor, and the key aspect of this interaction is the subcellular localization of the PS. PS can localize in mitochondria, lysosomes, endoplasmic reticulum, Golgi apparatus and plasma membranes. An explosion of investigation and explorations in the field of cell biology have elucidated many of the pathways that mammalian cells undergo when PS are delivered in tissue culture and subsequently illuminated. There is an acute stress response leading to changes in calcium and lipid metabolism and production of cytokines and stress proteins. Enzymes particularly, protein kinases, are activated and transcription factors are expressed. Many of the cellular responses are centered on mitochondria. These effects frequently lead to induction of apoptosis either by the mitochondrial pathway involving caspases and release of cytochrome c, or by pathways involving ceramide or death receptors. However, under certain circumstances cells subjected to PDT die by necrosis. Although there have been many reports of DNA damage caused by PDT, this is not thought to be an important cell-death pathway. This mechanistic research is expected to lead to optimization of PDT as a tumor treatment, and to rational selection of combination therapies that include PDT as a component.

Effect of Cell-Photosensitizer Binding and Cell Density on Microbial Photoinactivation

ABSTRACT Photodynamic therapy involves the use of nontoxic dyes called photosensitizers and visible light to produce reactive oxygen species and cell killing. It is being studied as an alternative method of killing pathogens in localized infections due to the increasing problem of multiantibiotic resistance. Although much has been learned about the mechanisms of microbial killing, there is still uncertainty about whether dyes must bind to and penetrate various classes of microbe in order to produce effective killing after illumination. In this report, we compare the interactions of three antimicrobial photosensitizers: rose bengal (RB), toluidine blue O (TBO), and a poly-l-lysine chlorin(e6) conjugate (pL-ce6) with representative members of three classes of pathogens; Escherichia coli (gram-negative bacteria), Staphylococcus aureus (gram-positive bacteria), Candida albicans (yeast). We compared fluence-dependent cell survival after illumination with the appropriate wavelengths of light before and after extracellular dye had been washed out and used three 10-fold dilutions of cell concentration. pL-ce6 was overall the most powerful photosensitizer, was equally effective with and without washing, and showed a strong dependence on cell concentration. TBO was less effective in all cases after washing, and the dependence on cell concentration was less pronounced. RB was ineffective after washing (except for S. aureus) but still showed a dependence on cell concentration. The overall order of susceptibility was S. aureus > E. coli > C. albicans, but C. albicans cells were 10 to 50 times bigger than the bacteria. We conclude that the number and mass of the cells compete both for available dye binding and for extracellularly generated reactive oxygen species.

Chitosan preparations for wounds and burns: antimicrobial and wound-healing effects

Since its discovery approximately 200 years ago, chitosan, as a cationic natural polymer, has been widely used as a topical dressing in wound management owing to its hemostatic, stimulation of healing, antimicrobial, nontoxic, biocompatible and biodegradable properties. This article covers the antimicrobial and wound-healing effects of chitosan, as well as its derivatives and complexes, and its use as a vehicle to deliver biopharmaceuticals, antimicrobials and growth factors into tissue. Studies covering applications of chitosan in wounds and burns can be classified into in vitro, animal and clinical studies. Chitosan preparations are classified into native chitosan, chitosan formulations, complexes and derivatives with other substances. Chitosan can be used to prevent or treat wound and burn infections not only because of its intrinsic antimicrobial properties, but also by virtue of its ability to deliver extrinsic antimicrobial agents to wounds and burns. It can also be used as a slow-release drug-delivery vehicle for growth factors to improve wound healing. The large number of publications in this area suggests that chitosan will continue to be an important agent in the management of wounds and burns.

Mechanisms in photodynamic therapy: Part three—Photosensitizer pharmacokinetics, biodistribution, tumor localization and modes of tumor destruction

Photodynamic therapy (PDT) has been known for over a hundred years, but is only now becoming widely used. Originally developed as cancer therapy, some of its most successful applications are for non-malignant disease. The majority of mechanistic research into PDT, however, is still directed towards anti-cancer applications. In the final part of series of three reviews, we will cover the possible reasons for the well-known tumor localizing properties of photosensitizers (PS). When PS are injected into the bloodstream they bind to various serum proteins and this can affect their phamacokinetics and biodistribution. Different PS can have very different pharmacokinetics and this can directly affect the illumination parameters. Intravenously injected PS undergo a transition from being bound to serum proteins, then bound to endothelial cells, then bound to the adventitia of the vessels, then bound either to the extracellular matrix or to the cells within the tumor, and finally to being cleared from the tumor by lymphatics or blood vessels, and excreted either by the kidneys or the liver. The effect of PDT on the tumor largely depends at which stage of this continuous process light is delivered. The anti-tumor effects of PDT are divided into three main mechanisms. Powerful anti-vascular effects can lead to thrombosis and hemorrhage in tumor blood vessels that subsequently lead to tumor death via deprivation of oxygen and nutrients. Direct tumor cell death by apoptosis or necrosis can occur if the PS has been allowed to be taken up by tumor cells. Finally the acute inflammation and release of cytokines and stress response proteins induced in the tumor by PDT can lead to an influx of leukocytes that can both contribute to tumor destruction as well as to stimulate the immune system to recognize and destroy tumor cells even at distant locations.