Tayyaba Hasan

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.

Imaging and Photodynamic Therapy: Mechanisms, Monitoring, and Optimization

The purpose of this review is to present the current state of the role of imaging in photodynamic therapy (PDT). In order for the reader to fully appreciate the context of the discussions embodied in this article we begin with an overview of the PDT process, starting with a brief historical perspective followed by detailed discussions of specific applications of imaging in PDT. Each section starts with an overview of the specific topic and, where appropriate, ends with summary and future directions. The review closes with the authors’ perspective of the areas of future emphasis and promise. The basic premise of this review is that a combination of imaging and PDT will provide improved research and therapeutic strategies.

Mechanisms of Action of Photodynamic Therapy with Verteporfin for the Treatment of Age-Related Macular Degeneration ☆ ☆☆

Age-related macular degeneration, especially the neovascular form of the disease, is the leading cause of blindness in elderly people in developed countries. Thermal photocoagulation is still the preferred treatment for choroidal neovascularization that does not involve the fovea, but it is suitable for only a small number of patients and it can lead to immediate loss of visual acuity. Photodynamic therapy with use of photochemical light activation of verteporfin as a photosensitizer (verteporfin therapy) has been shown to be effective in treating vascularized tumors, and its potential to treat other conditions involving neovascularization has also been suggested. Preclinical and clinical studies have indicated that verteporfin therapy can be used to treat choroidal neovascularization secondary to age-related macular degeneration effectively and safely. Selective occlusion of choroidal neovasculature by this therapy causes minimal damage to the neurosensory retina and, therefore, does not induce loss of visual acuity. This benefit allows verteporfin therapy to be used in the large proportion of patients who are not eligible for treatment by laser photocoagulation. The mechanistic aspects of the mode of action of light-activated verteporfin are described in this review.

Development and Applications of Photo-triggered Theranostic Agents

Theranostics, the fusion of therapy and diagnostics for optimizing efficacy and safety of therapeutic regimes, is a growing field that is paving the way towards the goal of personalized medicine for the benefit of patients. The use of light as a remote-activation mechanism for drug delivery has received increased attention due to its advantages in highly specific spatial and temporal control of compound release. Photo-triggered theranostic constructs could facilitate an entirely new category of clinical solutions which permit early recognition of the disease by enhancing contrast in various imaging modalities followed by the tailored guidance of therapy. Finally, such theranostic agents could aid imaging modalities in monitoring response to therapy. This article reviews recent developments in the use of light-triggered theranostic agents for simultaneous imaging and photoactivation of therapeutic agents. Specifically, we discuss recent developments in the use of theranostic agents for photodynamic-, photothermal- or photo-triggered chemotherapy for several diseases.

Vascular Targeting in Photodynamic Occlusion of Subretinal Vessels

PURPOSE To evaluate the potential of photodynamic therapy (PDT) using benzoporphyrin derivative (BPD) for occlusion of subretinal neovascular membranes, the authors studied efficiency and collateral damage of PDT-induced photothrombosis in the rabbit choriocapillary layer. METHOD Benzoporphyrin derivative, a new photosensitizer, currently in clinical trials for tumor therapy, was used. Low-density lipoprotein served as a carrier to enhance selective targeting of vascular endothelial cells. RESULTS Complete choriocapillary occlusion was achieved at a BPD dose of 2 mg/kg and a radiant exposure as low as 10 J/cm2. When PDT was performed 3 hours after BPD application, damage to the neural retina was minimal. Only inner photoreceptor segments showed mitochondrial swelling probably secondary to choroidal ischemia. Bruchs membrane remained intact. Retinal pigment epithelium was invariably damaged as seen with other photosensitizers. CONCLUSION Compared with photocoagulation BPD-PDT allows endothelial-bound intraluminal photothrombosis, sparing important structures such as neural retina and Bruchs membrane. It may thus provide a more selective treatment of juxtafoveal and subfoveal neovascular membranes.

Rapid Control of Wound Infections by Targeted Photodynamic Therapy Monitored by In Vivo Bioluminescence Imaging

The worldwide rise in antibiotic resistance necessitates the development of novel antimicrobial strategies. In this study we report on the first use of a photochemical approach to destroy bacteria infecting a wound in an animal model. Following topical application, a targeted polycationic photosensitizer conjugate between poly‐l‐lysine and chlorine6 penetrated the Gram (−) outer bacterial membrane, and subsequent activation with 660 nm laser light rapidly killed Escherichia coli infecting excisional wounds in mice. To facilitate real‐time monitoring of infection, we used bacteria that expressed the lux operon from Photorhabdus luminescens; these cells emitted a bioluminescent signal that allowed the infection to be rapidly quantified, using a low‐light imaging system. There was a light‐dose dependent loss of luminescence in the wound treated with conjugate and light, not seen in untreated wounds. Treated wounds healed as well as control wounds, showing that the photodynamic treatment did not damage the host tissue. Our study points to the possible use of this methodology in the rapid control of wounds and other localized infections.

Intracellular drug delivery by poly(lactic-co-glycolic acid) nanoparticles, revisited

We reexamined the cellular drug delivery mechanism by poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) to determine their utility and limitations as an intracellular drug delivery system. First, we prepared PLGA NPs which physically encapsulated Nile red (a hydrophobic fluorescent dye), in accordance with the usual procedure for labeling PLGA NPs, incubated them with mesothelial cells, and observed an increase in the intracellular fluorescence. We then prepared NPs from PLGA chemically conjugated to a fluorescent dye and observed their uptake by the mesothelial cells using confocal microscopy. We also used coherent anti-Stokes Raman scattering (CARS) microscopy to image cellular uptake of unlabeled PLGA NPs. Results of this study coherently suggest that PLGA NPs (i) are not readily taken up by cells, but (ii) deliver the payload to cells by extracellular drug release and/or direct drug transfer to contacting cells, which are contrasted with the prevalent view. From this alternative standpoint, we analyzed cytotoxicities of doxorubicin and paclitaxel delivered by PLGA NPs and compared with those of free drugs. Finally, we revisit previous findings in the literature and discuss potential strategies to achieve efficient drug delivery to the target tissues using PLGA NPs.

PHOTOPHYSICAL AND PHOTOSENSITIZING PROPERTIES OF BENZOPORPHYRIN DERIVATIVE MONOACID RING A (BPD‐MA)*

The photophysical properties of benzoporphyrin derivative monoacid ring A (BPD‐MA), a second‐generation photosensitizer currently in phase II clinical trials, were investigated in homogeneous solution. Absorption, fluorescence, triplet‐state, singlet oxygen (O2(1Δg)) sensitization studies and photobleaching experiments are reported. The ground state of this chlorin‐type molecule shows a strong absorbance in the red (λ≈ 688 nm, ɛ≈ 33 000 M−1 cm−1 in organic solvents). For the singlet excited state the following data were determined in methanol: energy level, Es= 42.1 kcal mol−1, lifetime, Φf= 5.2 ns and fluorescence quantum yield, Φf= 0.05 in air‐saturated solution. The triplet state of BPD‐MA has a lifetime, τf >. 25 ns, an energy level, ET= 26.9 kcal mol−1 and the molar absorption coefficient is ɛT= 26 650 M−1 cm−1 at 720 nm. A dramatic effect of oxygen on the fluorescence (φf) and intersystem crossing (φT) quantum yields has been observed. The BPD‐MA presents rather high triplet (φT= 0.68 under N2‐saturated conditions) and singlet oxygen (φΔ= 0.78) quantum yields. On the other hand, the presence of oxygen does not significantly modify the photobleaching of this photostable compound, the photodegradation quantum yield (φPb) of which was found to be on the order of 5 × 10−5 in organic solvents.

A three-dimensional in vitro ovarian cancer coculture model using a high-throughput cell patterning platform

In vitro 3D cancer models that provide a more accurate representation of disease in vivo are urgently needed to improve our understanding of cancer pathology and to develop better cancer therapies. However, development of 3D models that are based on manual ejection of cells from micropipettes suffer from inherent limitations such as poor control over cell density, limited repeatability, low throughput, and, in the case of coculture models, lack of reproducible control over spatial distance between cell types (e.g., cancer and stromal cells). In this study, we build on a recently introduced 3D model in which human ovarian cancer (OVCAR-5) cells overlaid on Matrigel™ spontaneously form multicellular acini. We introduce a high-throughput automated cell printing system to bioprint a 3D coculture model using cancer cells and normal fi broblasts micropatterned on Matrigel™ . Two cell types were patterned within a spatially controlled microenvironment (e.g., cell density, cell-cell distance) in a high-throughput and reproducible manner; both cell types remained viable during printing and continued to proliferate following patterning. This approach enables the miniaturization of an established macro-scale 3D culture model and would allow systematic investigation into the multiple unknown regulatory feedback mechanisms between tumor and stromal cells and provide a tool for high-throughput drug screening.

Effects of growth phase and extracellular slime on photodynamic inactivation of gram-positive pathogenic bacteria.

ABSTRACT The emergence of antibiotic resistance among pathogenic bacteria has led to efforts to find alternative antimicrobial therapeutics to which bacteria will not be easily able to develop resistance. One of these may be the combination of nontoxic dyes (photosensitizers [PS]) and visible light, known as photodynamic therapy, and we have reported its use to treat localized infections in animal models. While it is known that gram-positive species are generally susceptible to photodynamic inactivation (PDI), the factors that govern variation in degrees of killing are unknown. We used isogenic pairs of wild-type and transposon mutants deficient in capsular polysaccharide and slime production generated from Staphylococcus epidermidis and Staphylococcus aureus to examine the effects of extracellular slime on susceptibility to PDI mediated by two cationic PS (a polylysine-chlorine6 conjugate, pL-ce6, and methylene blue [MB]) and an anionic molecule, free ce6, and subsequent exposure to 665-nm light at 0 to 40 J/cm2. Free ce6 gave more killing of mutant strains than wild type, despite the latter taking up more PS. Log-phase cultures were killed more than stationary-phase cultures, and this correlated with increased uptake. The cationic pL-ce6 and MB gave similar uptakes and killing despite a 50-fold difference in incubation concentration. Differences in susceptibility between strains and between growth phases observed with free ce6 largely disappeared with the cationic compounds despite significant differences in uptake. These data suggest that slime production and stationary phase can be obstacles against PDI for gram-positive bacteria but that these obstacles can be overcome by using cationic PS.