Lothar Lilge

Silicon nanoparticles produced by femtosecond laser ablation in water as novel contamination-free photosensitizers

We report the synthesis of novel inorganic contamination-free photosensitizers based on colloidal silicon nanoparticles prepared by laser ablation in pure deionized water. We show that such nanoparticles are capable of generating singlet oxygen ((1)O(2)) under laser irradiation with a yield estimated at 10% of that of photofrin, which makes them a potential candidate for therapeutics, antiseptics, or disinfectants. We also discuss a model of (1)O(2) generation and the possibility for optimizing its release. Potential advantages of such novel inorganic photosensitizers include stable and nonphotobleaching (1)O(2) release, easy removal, and low dark toxicity.

Photodynamic Therapy of Vertebral Metastases: Evaluating Tumor-to-Neural Tissue Uptake of BPD-MA and ALA-PpIX in a Murine Model of Metastatic Human Breast Carcinoma †

Photodynamic therapy has been successfully applied to numerous cancers. Its potential to treat cancer metastases in the spine has been demonstrated previously in a preclinical animal model. The aim of this study was to test two photosensitizers, benzoporphyrin‐derivative monoacid ring A (BPD‐MA) and by 5‐aminolevulinic acid (5‐ALA)‐induced protoporphyrin IX (PpIX), for their potential use to treat bony metastases. The difference in photosensitizer concentration in the spinal cord and the surrounding tumor‐bearing vertebrae was of particular interest to assess the risk of potential collateral damage to the spinal cord. Vertebral metastases in a rat model were generated by intracardiac injection of human breast cancer cells. When tumor growth was confirmed, photosensitizers were injected systemically and the animals were euthanized at different time points. The following tissues were harvested: liver, kidney, ovaries, appendicular bone, spinal cord and lumbar vertebrae. Photosensitizer tissue concentration of BPD‐MA or PpIX was determined by fluorescence spectrophotometry. In contrast to BPD‐MA, ALA‐PpIX did not demonstrate an appreciable difference in the uptake ratio in tumor‐bearing vertebrae compared to spinal cord. The highest ratio for BPD‐MA concentration was found 15u2003min after injection, which can be recommended for therapy in this model.

Design and performance of thin cylindrical diffusers created in Ge-doped multimode optical fibers.

Cylindrical fiber diffusers have become common tools for various medical therapies. However, their large outer diameters and short lengths restrict their clinical application in some newly developed light therapies. Here, a 250-microm outer-diameter diffuser with an active length that exceeds 5 cm is presented. Diffusers are created in photosensitive optical fibers with outer cladding diameters of 140 microm by use of a structured beam from an excimer laser. Predetermined emission profiles can be achieved. Photometric characteristics, including longitudinal, polar, and azimuthal emission diagrams, were determined by use of a goniometer to assess the light-emission performance of the diffuser. Longitudinal isotropy of better than +/- 10% was achieved. Polar and azimuthal emissions were within +/- 15% of those of an ideal linear Lambertian emitter. Polar uniformity could be improved by an insignificant increase in the outer diameter by use of a diffusing recoating compound. The residual leakage of light at the distal end of the diffuser can be as little as 1%. Other physical parameters tested include minimal bending radius after recoating (< 5 mm) and maximum power handling (> 1.0W cm(-1)). All materials employed were biocompatible.

Optical spectroscopy of the breast in premenopausal women reveals tissue variation with changes in age and parity.

PURPOSEnVariations during breast tissue development can influence later breast cancer risk. In particular, prolonged nulliparity increases risk. The authors used optical spectroscopy to compare breast tissue in 115 nulliparous women aged 31-40 (group 2) to 140 nulliparous women aged 18-21 (group 1), and also to 36 parous women aged 31-40 (group 3), and to evaluate the relationship between IGF-1 and optical breast tissue properties. IGF-1 has been linked in particular to premenopausal breast cancer.nnnMETHODSnThe authors measured the transmission spectra from 625 to 1050 nm wavelengths in each breast and determined regions of interindividual variation using principal components analysis. Spectral differences represent variation in lipid, water, oxyhemoglobin, deoxyhemoglobin, and collagen content. Group differences and the relationship with IGF-1 were estimated by linear regression after adjustment for multiple factors including height, weight, ethnicity, hormonal contraceptive use, and days since last menstrual period.nnnRESULTSnPrincipal component 3 scores were more negative in the older nulliparous women compared to either younger nulliparous women or to parous women of the same age (beta = -0.16, p = 0.008 for group 2 vs group 1 and beta = 0.51, p = 0.03 for group 3 vs group 2). These differences appear to indicate increased deoxyhemoglobin relative to oxyhemoglobin content in the tissue of the older, nulliparous premenopausal women compared to the other groups, which may be an indicator of proportionally increased proliferative tissue. Principal component 4 also differed between older and younger nulliparous women (beta = 0.08, p = 0.02 for group 2 vs. group 1) and was negatively associated with IGF-1 in younger women (beta = -0.0004, p = 0.03) and positively associated with IGF-1 in older women (beta = 0.001, p = 0.004).nnnCONCLUSIONSnOptical spectroscopy may be useful to identify breast tissue at increased risk of cancer development and track changes over time, particularly in young women where exposure to radiation is of particular concern. Additional work is needed to confirm the observed breast tissue differences and to determine the specific tissue chromophore changes with age and parity.

A Ratiometric Fluorescence Imaging System for Surgical Guidance

Correspondence should be addressed to Brian C. Wilson, wilson@uhnres.utoronto.caReceived 3 March 2008; Accepted 28 June 2008Recommended by Stoyan TanevA 3-chip CCD imaging system has been developed for quantitative in vivo fluorescence imaging. This incorporates a ratiometricalgorithm to correct for the effects of tissue optical absorption and scattering, imaging “geometry” and tissue autofluorescencebackground. The performance was characterized, and the algorithm was validated in tissue-simulating optical phantoms forquantitative measurement of the fluorescent molecule protoporphyrin IX (PpIX). The technical feasibility to use this system forfluorescence-guided surgical resection of malignant brain tumor tissue was assessed in an animal model in which PpIX was inducedexogenously in the tumor cells by systemic administration of aminolevulinic acid (ALA).Copyright

Targeting non-small cell lung cancer by novel TLD-1433-mediated photodynamic therapy (Conference Presentation)

Background: The majority of cancers upregulate their transferrin receptor (Tf-R) to satisfy their higher Fe3+ requirements for proliferation. TLD-1433 can bind to transferrin to form Rutherrin, which is a promising photosensitizer with stable chemical structure and higher tissue selectivity. nMethods: To investigate the effect of Rutherrin®-mediated photodynamic treatment (PDT), we used non-small lung cancer cell lines H2170, A549, and H460. Subcutaneous tumors were treated with Rutherrin-mediated PDT, 4hrs post intravenous administration. The treatment parameters10 mg/kg Rutherrin and 600 Jcm-2 808 nm radiation. In an orthotopic A549 tumor model, the presence of tumor after inoculation in lungs was confirmed by microCT. Tissue samples were collected for Inductively Coupled Mass Spectrometry to quantify the Rutherrin concentrations via a Ru isotope in tumor and normal lung tissue. nResults: Evaluation of TfR expression by flow cytometric and western blotting showed that almost all cancer cells express TfR. In in-vitro cytotoxicity assay, all cancer cell lines showed high cell kill by PDT at 100nM Rutherrin concentrations. In the subcutaneous tumor model, PDT after Rutherrin injection significantly inhibited the tumor growth and histopathology showed extensive necrosis at 24 hrs, which was confirmed with lowered Ki67 staining. In an orthotopic model, the lung lobe with tumor retained more Rutherrin than the contralateral lung, showing specific tumor uptake.nConclusion: These results support the hypothesis that safe and efficient Rutherrin-mediated PDT is feasible due to improved photosensitizer localization to lung tumors tissue. Selective irradiation of the cancer lesions by strategic placement of the light source remains a requirement.

Two-photon luminescence lifetime imaging microscopy (LIM) to follow up cell metabolism and oxygen consumption during theranostic applications

A common property during tumor development is altered energy metabolism, which could lead to a switch from oxidative phosphorylation and glycolysis. The impact of this switch for theranostic applications could be significant. Interestingly altered metabolism could be correlated with a change in the fluorescence lifetimes of both NAD(P)H and FAD. However, as observed in a variety of investigations, the situation is complex and the result is influenced by parameters like oxidative stress, pH or viscosity. Besides metabolism, oxygen levels and consumption has to be taken into account in order to understand treatment responses. For this, correlated imaging of phosphorescence and fluorescence lifetime parameters has been investigated by us and used to observe metabolic markers simultaneously with oxygen concentrations. The technique is based on time correlated single photon counting to detect the fluorescence lifetime of NAD(P)H and FAD by FLIM and the phosphorescence lifetime of newly developed phosphors and photosensitizers by PLIM. For this, the photosensitizer TLD1433 from Theralase, which is based on a ruthenium (II) coordination complex, was used. TLD1433 which acts as a redox indicator was mainly found in cytoplasmatic organelles. The most important observation was that TLD1433 can be used as a phosphor to follow up local oxygen concentration and consumption during photodynamic therapy. Oxygen consumption was accompanied by a change in cell metabolism, observed by simultaneous FLIM/PLIM. The combination of autofluorescence-FLIM and phosphor-PLIM in luminescence lifetime microscopy provides new insights in light induced reactions.

Personalizing cytotoxic dose for Ru(II) coordination complex mediated photodynamic therapy in nonmuscle invasive bladder cancer (Conference Presentation)

Non-muscle invasive bladder cancer remains one of the costliest cancer to treat, and while a cystectomy will reduce a patient’s risk of developing metastatic disease it reduced the patient’s quality of life. While Photofrin mediate Photodynamic Therapy was approved already in 1993, poor control over the photon density and drug accumulation in of target tissue resulted in overdosing the bladder muscle layers, causing permanent volume shrinkage and incontinence. nnFor an ongoing Phase Ib clinical trial, evaluating the safety of TLD1433 ([Ru(II)(4,4-dimethyl-2,2-bipyridine(dmb))2(2-(2,2:5,2-terthiophene)-imidazo[4,5-f][1,10]phenanthroline)]2+) a Ru(II) coordination complex, significant deviations from the previous studies are implemented. The photosensitizer is instilled, to reduce the sensitization of the muscle layer, 525 nm light is used to limit the light penetration into the bladder and the photon density is measured in each patient at up to 12 positions. nnImproved tumour selectivity is provided by this photosensitizer as it is block by the Urothelium from entering healthy tissue, whereas it enters tumour cells, supposing via the transferrin receptor, as demonstrated in in-vitro and in vivo studies. The Ru(II) coordination complex stains tissues where the urothelium is damaged very strongly in an orange-rust colour, visible under white light illumination.nnPreclinical in vivo studies showed the destruction of tumours up to 1 mm in depth following 1 hr of drug instillation, followed by 3 washes and the delivery of 90 Jcm-2 of 525nm light in the wistar rat Ay-27 tumour model. Histology showed very limited muscle damage and in general intact urothelium layers, with a moderate infiltration of macrophages.nnTo achieve the prescribed target radiant exposure of 90 Jcm-2, independent of the bladder tissue diffuse reflectivity and shape, an optical dosimetry system was developed which can be deployed via a cystoscope. The optical dose monitoring device allows the treating physician to adjust the source position to achieve the target optical dose for the 12 sensor positions. The optical radiation was delivered via a 0.8 mm diameter spherical diffuser at up to 2.5 W power.nnThe attainable photon density and the anticipated PDT dose are simulated for each patient using a photon propagation engine and the patient’s anatomical information. During these simulations, a range of tissue optical properties is simulated and compared to the initial photon density measurements to advise the physician further about the ability to achieve a homogenous illumination in each patient. nnThe multiplication factor of the irradiance inside the bladder, calculated based on the spherical volume equivalent size of the bladder, the delivered power and measured irradiance, varied between patients (from 1.1 to 2.5) and also over the course of the treatment. The changes over the course of the treatment were predominantly due to light diffusing proteinaceous material floating in the bladder. nnExpanding prior work by the Rotterdam group on light propagation in the bladder we determined that the tissue albedo varied from 0.87 to 0.92 for 525 nm in this patient population. The estimated average effective attenuation coefficient for this population was approximately 1 mm adequate for the treatment of non-muscle invasive bladder cancer after transurethral resection of the large tumours. nnMonte Carlo modelling demonstrated that the fluence as function of depth into the tissue is determined by the exposure of bladder wall elements to the remaining bladder surface and can vary by factor of more than 2 even when assuming homogenous tissue optical properties across the bladder wall surface.nnThese studies and analysis demonstrate the need for accurate light dosimetry in hollow organs when variations in the tissue albedo and the shape of the organ can influence the photodynamic dose significantly.

Ru(II) complex mediated PDT for bladder cancer, Biology and dosimetry

Metal-based photo sensitizers are of interest as their absorption and chemical binding properties can be modified via the use of different ligands. Ru2+ based photo sensitizers are known to be effective photodynamic therapy (PDT) agents against bacteria, whereas use for oncological indications in vivo has not been demonstrated with the same level of evidence. We present data showing that premixing the Ru2+-complex TLD14331,2 ([Ru(II)(4,4-dimethyl-2,2-bipyridine(dmb))2(2-(2,2:5,2-terthiophene)-imidazo[4,5-f][1,10]phenanthroline)]2+) with transferrin increases the molar extinction coefficient, including longer activation wavelengths, reduces photobleaching rates, reduces the toxicity of the complex and improving overall PDT efficacy demonstrated in Human (HT 1376) and rat (AY27) bladder cancer cells. As the transferrin receptor is upregulated in most malignancies, premixing of the Ru2+ complex with transferrin converts the active pharmaceutical ingredient TLD1433 into a drug of potentially considerable clinical utility.

CHAPTER 19:Pre-Clinical Models for Glioma Photodynamic Therapy

The efficacy of photodynamic therapy (PDT) for the treatment of primary and recurrent gliomas has been evaluated in a fair number of clinical trials over the past two decades. While some studies showed an effect, it is evident that PDT as a standalone or adjuvant to standard therapy has highly variable efficacy and bears morbidity risks for some patients, preventing its broader adoption in the clinic. Cofactors as causes for the observed efficacy variance and strategies to improve PDT efficacy need to be determined in preclinical models, and the same applies to dosimetry concepts and response monitoring strategies. However, the use of small murine models poses in itself challenges for PDT research, such as the intracranial dimensions between the tumor and eloquent areas of the brain being small compared to the light and hence PDT dose gradient. Here, we present current work pertaining to the use of preclinical models for the PDT-mediated destruction of gliomas, including the biological relevance of commonly used glioma cell lines, the means to modify protoporphyrin IX (PpIX) synthesis in vivo and the tissue responsivity to PpIX-mediated intracranial PDT, as well as novel magnetic resonance imaging-based approaches for predicting tissue response. Employing the appropriate preclinical models has the potential for rapid hypothesis generation and testing in order to design improved clinical studies in the future that will be needed for demonstrating improved efficacy in the vast majority of patient if PDT is to become a first-line therapy in neurosurgery.