Mark T. Jarvi

Photodynamic molecular beacon as an activatable photosensitizer based on protease-controlled singlet oxygen quenching and activation

Molecular beacons are FRET-based target-activatable probes. They offer control of fluorescence emission in response to specific cancer targets, thus are useful tools for in vivo cancer imaging. Photodynamic therapy (PDT) is a cell-killing process by light activation of a photosensitizer (PS) in the presence of oxygen. The key cytotoxic agent is singlet oxygen (1O2). By combining these two principles (FRET and PDT), we have introduced a concept of photodynamic molecular beacons (PMB) for controlling the PSs ability to generate 1O2 and, ultimately, for controlling its PDT activity. The PMB comprises a disease-specific linker, a PS, and a 1O2 quencher, so that the PSs photoactivity is silenced until the linker interacts with a target molecule, such as a tumor-associated protease. Here, we report the full implementation of this concept by synthesizing a matrix metalloproteinase-7 (MMP7)-triggered PMB and achieving not only MMP7-triggered production of 1O2 in solution but also MMP7-mediated photodynamic cytotoxicity in cancer cells. Preliminary in vivo studies also reveal the MMP7-activated PDT efficacy of this PMB. This study validates the core principle of the PMB concept that selective PDT-induced cell death can be achieved by exerting precise control of the PSs ability to produce 1O2 by responding to specific cancer-associated biomarkers. Thus, PDT selectivity will no longer depend solely on how selectively the PS can be delivered to cancer cells. Rather, it will depend on how selective a biomarker is to cancer cells, and how selective the interaction of PMB is to this biomarker.

Optimized speckle variance OCT imaging of microvasculature

We optimize speckle variance optical coherence tomography (svOCT) imaging of microvasculature in high and low bulk tissue motion scenarios. To achieve a significant level of image contrast, frame rates must be optimized such that tissue displacement between frames is less than the beam radius. We demonstrate that higher accuracy estimates of speckle variance can enhance the detection of capillaries. These findings are illustrated in vivo by imaging the dorsal window chamber model (low bulk motion). We also show svOCT imaging of the nonstabilized finger (high bulk motion), using optimized imaging parameters, demonstrating better vessel detection than Doppler OCT.

FRET Quenching of Photosensitizer Singlet Oxygen Generation

The development of activatable photodynamic therapy (PDT) has demonstrated a utility for effective photosensitizer quenchers. However, little is known quantitatively about Forster resonance energy transfer (FRET) quenching of photosensitizers, even though these quenchers are versatile and readily available. To characterize FRET deactivation of singlet oxygen generation, we attached various quenchers to the photosensitizer pyropheophorbide-alpha (Pyro) using a lysine linker. The linker did not induce major changes in the properties of the photosensitizer. Absorbance and emission wavelength maxima of the quenched constructs remained constant, suggesting that quenching by ground-state complex formation was minimal. All quenchers sharing moderate spectral overlap with the fluorescence emission of Pyro (J > or = 5.1 x 10(13) M(-1) cm(-1) nm4) quenched over 90% of the singlet oxygen, and quenchers with weaker spectral overlap displayed minimal quenching. A self-quenched double Pyro construct exhibited intermediate quenching. Consistent with a FRET deactivation mechanism, extension of the linker to a 10 residue polyproline peptide resulted in only the quenchers with spectral overlap almost 2 orders of magnitude higher (J > or = 3.7 x 10(15) M(-1) cm(-1) nm4) maintaining high quenching efficiency. Overall, there was good correlation (0.98) between fluorescence quenching and singlet oxygen quenching, implying that fluorescence intensity can be a convenient indicator for the singlet oxygen production status of activatable photosensitizers. Uniform singlet oxygen luminescence lifetimes of the compounds, along with minimal triplet state transient absorption were consistent with quenchers primarily deactivating the photosensitizer excited singlet state. In vitro, cells treated with well-quenched constructs demonstrated greatly reduced PDT induced toxicity, indicating that FRET-based quenchers can provide a level of quenching useful for future biological applications. The presented findings show that FRET-based quenchers can potently decrease singlet oxygen production and therefore be used to facilitate the rational design of activatable photosensitizers.

Correlation between cell viability and cumulative singlet oxygen luminescence from protoporphyrin IX in varying subcellular localizations

Photodynamic therapy (PDT) can be targeted toward different subcellular localizations and it is widely believed different subcellular targets vary in their sensitivity to photobiological damage. In this study, PDT-generated near-infrared singlet oxygen (1O2) luminescence was measured along with cell viability under two different incubation protocols: 5- aminolevulinic acid (ALA) endogenously-induced protoporphyrin IX (PpIX) and exogenous PpIX, at different incubation times. Confocal fluorescence microscopy indicated that ALA-induced PpIX (2 h) localized in the mitochondria, whereas exogenous PpIX (1 h) mainly localized to the plasma membrane. Cell viability was determined at several time points during PDT treatments using colony-forming assays, and the surviving fraction correlated well with cumulative 1O2 luminescence counts under both incubation protocols. Preliminary results indicate the plasma membrane is less sensitive to PDT-generated 1O2 than the mitochondria.

A Monte Carlo model of detected singlet oxygen luminescence and photosensitizer fluorescence during ALA-PDT of skin

Singlet Oxygen (1O2) Luminescence Dosimetry (SOLD) and fluorescence photobleaching are being investigated as dosimetric tools for clinical PDT. Both have been applied during superficial ALA-PDT of normal skin and skin cancers. The interpretation of fluorescence and SOLD data is complicated by the non-uniform distribution and bleaching of PpIX and the absorption and scattering of light in the skin. The aim of the present work was to tackle these challenges using Monte Carlo (MC) simulations. Skin was modeled as a three-layer semi-infinite medium with uniform optical properties in each layer. The initial depth-dependent distribution of PpIX was an exponential decay and, after the delivery of each treatment fluence increment, standard photochemical reaction kinetics were used to update the distribution of sensitizer and reacted singlet oxygen. Oxygen depletion due to photochemical consumption or vascular shutdown was also incorporated in the model. The adjoint method was applied to calculate the PpIX fluorescence and 1270 nm singlet oxygen luminescence reaching the skin surface in each time increment. The time-resolved evolution of the fluorescence and cumulative SOLD signals during treatment were compared to the time-resolved volume-averaged distribution of reacted singlet oxygen in the dermis layer for typical clinical PDT conditions. Approximate linear relationships were observed over most of the treatment time.

In vitro influence of hypoxia on bioluminescence imaging in brain tumor cells

Bioluminescence Imaging (BLI) has been employed as an imaging modality to identify and characterize fundamental processes related to cancer development and response at cellular and molecular levels. This technique is based on the reaction of luciferin with oxygen in the presence of luciferase and ATP. A major concern in this technique is that tumors are generally hypoxic, either constitutively and/or as a result of treatment, therefore the oxygen available for the bioluminescence reaction could possibly be reduced to limiting levels, and thus leading to underestimation of the actual number of luciferase-labeled cells during in vivo procedures. In this report, we present the initial in vitro results of the oxygen dependence of the bioluminescence signal in rat gliosarcoma 9L cells tagged with the luciferase gene (9Lluc cells). Bioluminescence photon emission from cells exposed to different oxygen tensions was detected by a sensitive CCD camera upon exposure to luciferin. The results showed that bioluminescence signal decreased at administered pO2 levels below about 5%, falling by approximately 50% at 0.2% pO2. Additional experiments showed that changes in BLI was due to the cell inability to maintain normal levels of ATP during the hypoxic period reducing the ATP concentration to limiting levels for BLI.