Light Fractionation Significantly Increases the Efficacy of Photodynamic Therapy Using BF-200 ALA in Normal Mouse Skin.

Background: Light fractionation significantly increases the efficacy of 5-aminolevulinic acid (ALA) based photodynamic therapy (PDT) using the nano-emulsion based gel formulation BF-200. PDT using BF-200 ALA has recently been clinically approved and is under investigation in several phase III trials for the treatment of actinic keratosis. This study is the first to compare BF-200 ALA with ALA in preclinical models.

Somatostatin analogues for receptor targeted photodynamic therapy.

Photodynamic therapy (PDT) is an established treatment modality, used mainly for anticancer therapy that relies on the interaction of photosensitizer, light and oxygen. For the treatment of pathologies in certain anatomical sites, improved targeting of the photosensitizer is necessary to prevent damage to healthy tissue. We report on a novel dual approach of targeted PDT (vascular and cellular targeting) utilizing the expression of neuropeptide somatostatin receptor (sst2) on tumor and neovascular-endothelial cells.

Topical photodynamic therapy using different porphyrin precursors leads to differences in vascular photosensitization and vascular damage in normal mouse skin.

Different distributions of hexyl aminolevulinate (HAL), aminolevulinic acid (ALA) and methyl aminolevulinate (MAL) in the superficial vasculature are not well studied but they are hypothesized to play an important role in topical photodynamic therapy (PDT). The colocalization of fluorescent CD31 and protoporphyrin IX (PpIX) was calculated using confocal microscopy of mouse skin sections to investigate the vascular distribution after topical application. Vascular damage leads to disruption of the normal endothelial adherens junction complex, of which CD144 is an integral component.

Intrinsic photosensitizer fluorescence measured using multi-diameter single-fiber spectroscopy in vivo.

Quantification of fluorescence in vivo is complicated by the influence of tissue optical properties on the collected fluorescence signal. When tissue optical properties in the measurement volume are quantified, one can obtain the intrinsic fluorescence, which equals the product of fluorophore absorption coefficient and quantum yield. We applied this method to in vivo single-fiber fluorescence spectroscopy measurements on mouse tongue, skin, liver, and oral squamous cell carcinoma, where we detected intrinsic fluorescence spectra of the photosensitizers chlorin e6 and Bremachlorin at t=[3,4.

Microscopic analysis of the localization of two chlorin-based photosensitizers in OSC19 tumors in the mouse oral cavity.

Background And Objective: The effect of photodynamic therapy (PDT) is dependent on the localization of photosensitizer in the treatment volume at the time of illumination. Investigation of photosensitizer pharmacokinetics in and around the treatment volume aids in determining the optimal drug light interval for PDT.

Materials And Methods: In this paper we have investigated the distribution of the photosensitizers chlorin e6 and Bremachlorin in the oral squamous cell carcinoma cell-line OSC19-Luc-Gfp in a tongue tumor, tumor boundary, invasive tumor boundary, and normal tongue tissue by the use of confocal microscopy of frozen sections.

Localization of liposomal mTHPC formulations within normal epithelium, dysplastic tissue, and carcinoma of oral epithelium in the 4NQO-carcinogenesis rat model.

Background And Objective: Foslip and Fospeg are liposomal formulations of the photosensitizer mTHPC (Foscan), which is used for photodynamic therapy (PDT) of malignancies. Literature suggests that liposomal mTHPC formulations have better properties and increased tumor uptake compared to Foscan. To investigate this, we used the 4NQO-induced carcinogen model to compare the localization of the different mTHPC formulations within normal, precancerous, and cancerous tissue.

Fluorescence localization and kinetics of mTHPC and liposomal formulations of mTHPC in the window-chamber tumor model.

Background And Objective: Foslip® and Fospeg® are liposomal formulations of the photosensitizer mTHPC, intended for use in Photodynamic Therapy (PDT) of malignancies. Foslip consists of mTHPC encapsulated in conventional liposomes, Fospeg consists of mTHPC encapsulated in pegylated liposomes. Possible differences in tumor fluorescence and vasculature kinetics between Foslip, Fospeg, and Foscan® were studied using the rat window-chamber model.

In vivo monitoring of protein-bound and free NADH during ischemia by nonlinear spectral imaging microscopy.

Nonlinear spectral imaging microscopy (NSIM) allows simultaneous morphological and spectroscopic investigation of intercellular events within living animals. In this study we used NSIM for in vivo time-lapse in-depth spectral imaging and monitoring of protein-bound and free reduced nicotinamide adenine dinucleotide (NADH) in mouse keratinocytes following total acute ischemia for 3.3 h at ~3 min time intervals.

In vivo quantification of photosensitizer fluorescence in the skin-fold observation chamber using dual-wavelength excitation and NIR imaging.

A major challenge in biomedical optics is the accurate quantification of in vivo fluorescence images. Fluorescence imaging is often used to determine the pharmacokinetics of photosensitizers used for photodynamic therapy. Often, however, this type of imaging does not take into account differences in and changes to tissue volume and optical properties of the tissue under interrogation.

Fractionated illumination at low fluence rate photodynamic therapy in mice.

Photodynamic therapy (PDT) for actinic field cancerization is effective but painful. Pain mechanisms remain unclear but fluence rate has been shown to be a critical factor. Lower fluence rates also utilize available oxygen more efficiently.

A telemetric light delivery system for metronomic photodynamic therapy (mPDT) in rats.

Light delivery and monitoring during photodynamic therapy (PDT) is often limited by the need for a physical link between the light source, detectors and the treatment volume. This paper reports on the first in vivo experiments performed with a fully implantable telemetric system, designed for a rat glioblastoma model. In this system, light delivery is performed using a solid state optode containing 2 LEDs, and 4 photodiodes which will be used to monitor light delivery in future experiments.

Monitoring interstitial m-THPC-PDT in vivo using fluorescence and reflectance spectroscopy.

Background And Objective: In order to understand the mechanisms of photodynamic therapy (PDT) it is important to monitor parameters during illumination that yield information on deposited PDT dose. The aim of this study is to investigate the possibility of monitoring implicit parameters, such as photobleaching, in addition to monitoring explicit parameters (fluence (rate), oxygenation, photosensitizer concentration) directly or indirectly. These parameters are monitored during PDT without interrupting the therapeutic illumination.

In vivo quantification of chromophore concentration using fluorescence differential path length spectroscopy.

We present an optical method based on fluorescence spectroscopy for measuring chromophore concentrations in vivo. Fluorescence differential path length spectroscopy (FPDS) determines chromophore concentration based on the fluorescence intensity corrected for absorption. The concentration of the photosensitizer m-THPC (Foscan) was studied in vivo in normal rat liver, which is highly vascularized and therefore highly absorbing.

Design and implementation of a sensitive high-resolution nonlinear spectral imaging microscope.

Live tissue nonlinear microscopy based on multiphoton autofluorescence and second harmonic emission originating from endogenous fluorophores and noncentrosymmetric-structured proteins is rapidly gaining interest in biomedical applications. The advantage of this technique includes high imaging penetration depth and minimal phototoxic effects on tissues. Because fluorescent dyes are not used, discrimination between different components within the tissue is challenging.

In vivo nonlinear spectral imaging microscopy of visible and ultraviolet irradiated hairless mouse skin tissues.

We demonstrate the capability of nonlinear spectral imaging microscopy (NSIM) in investigating ultraviolet and visible light induced effects on albino Skh:HR-1 hairless mouse skin non-invasively.

Microscopic localisation of protoporphyrin IX in normal mouse skin after topical application of 5-aminolevulinic acid or methyl 5-aminolevulinate.

Light fractionation does not enhance the response to photodynamic therapy (PDT) after topical methyl-aminolevulinate (MAL) application, whereas it is after topical 5-aminolevulinic acid (ALA). The differences in biophysical and biochemical characteristics between MAL and ALA may result in differences in localisation that cause the differences in response to PDT. We therefore investigated the spatial distribution of protoporphyrin IX (PpIX) fluorescence in normal mouse skin using fluorescence microscopy and correlated that with the PDT response histologically observed at 2.

Monitoring ALA-induced PpIX photodynamic therapy in the rat esophagus using fluorescence and reflectance spectroscopy.

The presence of phased protoporphyrin IX (PpIX) bleach kinetics has been shown to correlate with esophageal response to 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) in animal models. Here we confirm the existence of phased PpIX photobleaching by increasing the temporal resolution of the fluorescence measurements using the therapeutic illumination and long wavelength fluorescence detection. Furthermore fluorescence differential pathlength spectroscopy (FDPS) was incorporated to provide information on the effects of PpIX and tissue oxygenation distribution on the PpIX bleach kinetics during illumination.

Ex vivo quantification of mTHPC concentration in tissue: influence of chemical extraction on the optical properties.

A method for the quantification of the concentration of the photosensitizer meso-tetra(hydroxyphenyl) chlorin (mTHPC) in tissue samples is presented. The technique is an extension of a previously published method based on alkaline hydrolysis of tissue, using Solvable as a tissue solubilizer. mTHPC quantification was achieved by subsequent fluorescence spectroscopy.

Light fractionation does not enhance the efficacy of methyl 5-aminolevulinate mediated photodynamic therapy in normal mouse skin.

Previous work demonstrated that fractionated illumination using two fractions separated by a dark interval of 2 h, significantly enhanced the clinical efficacy of photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA). Considering the increasing clinical use of methyl 5-aminolevulinate (MAL) and the expected gain in efficacy by light fractionation we have investigated the response to MAL-PDT using a single and a two-fold illumination scheme and compared that with ALA-PDT. Our results show that fractionated illumination does not enhance the efficacy of PDT using MAL as it does using ALA despite the comparable fluorescence intensities at the end of the first light fraction and at the start of the second light fraction.

Increase in protoporphyrin IX after 5-aminolevulinic acid based photodynamic therapy is due to local re-synthesis.

Protoporphyrin IX (PpIX) fluorescence that is bleached during aminolevulinic acid (ALA) mediated photodynamic therapy (PDT) increases again in time after treatment. In the present study we investigated if this increase in PpIX fluorescence after illumination is the result of local re-synthesis or of systemic redistribution of PpIX. We studied the spatial distribution of PpIX after PDT with and without cooling using the skin-fold observation chamber model.

Spectrally resolved multiphoton imaging of in vivo and excised mouse skin tissues.

The deep tissue penetration and submicron spatial resolution of multiphoton microscopy and the high detection efficiency and nanometer spectral resolution of a spectrograph were utilized to record spectral images of the intrinsic emission of mouse skin tissues. Autofluorescence from both cellular and extracellular structures, second-harmonic signal from collagen, and a narrowband emission related to Raman scattering of collagen were detected. Visualization of the spectral images by wavelength-to-RGB color image conversion allowed us to identify and discriminate tissue structures such as epidermal keratinocytes, lipid-rich corneocytes, intercellular structures, hair follicles, collagen, elastin, and dermal cells.

Evidence for a bystander role of neutrophils in the response to systemic 5-aminolevulinic acid-based photodynamic therapy.

Background/purpose: A significant increase in the number of circulating and tumour neutrophils immediately after therapy was observed while investigating the increase in response of tissues to aminolevulinic acid-based photodynamic therapy (ALA-PDT) using a twofold illumination scheme with a prolonged dark interval. The action of (tumour) neutrophils is an important therapeutic adjunct to the deposition of singlet oxygen within the treatment volume, for many photosensitizers. It is not known if those phagocytes contribute to the improved outcome of ALA-PDT.