Soumya Mitra

Photobleaching kinetics of Photofrin in vivo and in multicell tumour spheroids indicate two simultaneous bleaching mechanisms

We present a detailed investigation of Photofrin photobleaching and photoproduct accumulation. Fisher rats were sensitized with 10 mg kg(-1) Photofrin and irradiated 24 h later with 514 nm light at 5 or 100 mW cm(-2). Fluorescence spectra were collected from the skin throughout treatment, and sensitizer bleaching and fluorescent photoproduct formation were quantified using spectral analysis. Photofrin bleaching was slightly more rapid at the higher irradiance under these conditions. However, accumulation of photoproduct was significantly enhanced at lower irradiance. To interpret these unexpected findings, we developed a new mathematical model in which reactions between singlet oxygen (1O2) and the photosensitizer and reactions between the sensitizer triplet and biological targets are both allowed to contribute to bleaching. Predictions of this model were tested in experiments performed on EMT6 spheroids sensitized with concentrations of 2.5, 10 and 30 microg mL(-1) Photofrin and subjected to PDT. Photofrin bleaching and photoproduct formation in these spheroids were measured using confocal fluorescence spectroscopy. In qualitative agreement with the mixed-mechanism model predictions, at the highest drug concentration Photofrin bleaching was more efficient via 1O2 reactions, while at the lowest concentration triplet reactions were more efficient. At all concentrations, photoproduct accumulation was greater under conditions of abundant oxygen.

Sensitivity of Candida albicans Germ Tubes and Biofilms to Photofrin-Mediated Phototoxicity

ABSTRACT Treatment of mucocutaneous and cutaneous Candida albicans infections with photosensitizing agents and light, termed photodynamic therapy (PDT), offers an alternative to conventional treatments. Initial studies using the clinically approved photosensitizer Photofrin demonstrated the susceptibility of C. albicans to its photodynamic effects. In the present study, we have further refined parameters for Photofrin-mediated photodynamic action against C. albicans and examined whether mechanisms commonly used by microorganisms to subvert either antimicrobial oxidative defenses or antimicrobial therapy, including biofilm formation, were operative. In buffer and defined medium, germ tubes preloaded with Photofrin retained their photosensitivity for up to 2 hours, indicating the absence of degradation or export of Photofrin by the organism. The addition of serum resulted in a gradual loss of photosensitivity over 2 hours. In contrast to an adaptive response by germ tubes to oxidative stress by hydrogen peroxide, there was no adaptive response to singlet oxygen-mediated stress by photodynamic action. C. albicans biofilms were sensitive to Photofrin-mediated phototoxicity in a dose-dependent manner. Finally, the metabolic activity of C. albicans biofilms following photodynamic insult was significantly lower than that of biofilms treated with amphotericin B for the same time period. These results demonstrate that several of the mechanisms microorganisms use to subvert either antimicrobial oxidative defenses or antimicrobial therapy are apparently not operative during Photofrin-mediated photodynamic treatment of C. albicans. These observations provide support and rationale for the continued investigation of PDT as an adjunctive, or possibly alternative, mode of therapy against cutaneous and mucocutaneous candidiasis.

Structural and molecular basis of the role of starch and sucrose in Streptococcus mutans biofilm development.

ABSTRACT The interaction of sucrose and starch with bacterial glucosyltransferases and human salivary amylase may enhance the pathogenic potential of Streptococcus mutans within biofilms by influencing the structural organization of the extracellular matrix and modulating the expression of genes involved in exopolysaccharide synthesis and specific sugar transport and two-component systems.

Photophysical Parameters, Photosensitizer Retention and Tissue Optical Properties Completely Account for the Higher Photodynamic Efficacy of meso‐Tetra‐Hydroxyphenyl‐Chlorin vs Photofrin¶

Meso‐tetra‐hydroxyphenyl‐chlorin (mTHPC) is one of the most potent photosensitizers currently available for clinical photodynamic therapy (PDT). However the reason or reasons for its high photodynamic efficacy remain(s) unresolved. To investigate the PDT efficacy of mTHPC vs Photofrin we use the knowledge of photophysical parameters extracted from the analysis of oxygen electrode measurements in spheroids to compute and compare their respective singlet oxygen (1O2 dose depositions. The electrode measurements indirectly report the bleaching kinetics of mTHPC and indicate that its photobleaching mechanism is consistent with 1O2‐mediated reactions. mTHPCs photodegradation via1O2 reactions is confirmed by a more direct evaluation of the spatially resolved fluorescence in confocal sections of intact spheroids during irradiation. The PDT efficacy comparisons establish that mTHPCs enhanced potency may be accounted for completely on the basis of its ability to sequester tightly in cells and its photophysical properties, in particular its higher extinction coefficient at a redshifted wavelength. We extend the efficacy comparison to include the influence of hemoglobin absorption of PDT treatment light and show that incorporating the influence of wavelength‐dependent light attenuation in tissue further contributes to significantly higher efficacy for mTHPC‐ vs Photofrin‐PDT.

A comprehensive mathematical model of microscopic dose deposition in photodynamic therapy

We have developed a comprehensive theoretical model for rigorously describing the spatial and temporal dynamics of oxygen (3O2) consumption and transport and microscopic photodynamic dose deposition during photodynamic therapy (PDT) in vivo. Previously published models have been improved by considering perfused vessels as a time-dependent 3O2 source and linking the 3O2 concentration in the vessel to that within the tissue through the Hill equation. The time-dependent photochemical 3O2 consumption rate incorporates sensitizer photobleaching effects and an experimentally determined initially nonuniform photosensitizer distribution. The axial transport of 3O2 is provided for in the capillaries and in the surrounding tissue. A self-sensitized singlet oxygen (1O2)-mediated bleaching mechanism and the measured, initially nonuniform distribution of mesotetrahydroxyphenyl chlorin at 3 h after intravascular administration were used to demonstrate the capabilities of the model. Time-evolved distributions of 3O2 concentration were obtained by numerically solving two-dimensional diffusion-with-reaction equations both in the capillary and the adjacent tissue. Using experimentally established physiological and photophysical parameters, the mathematical model allows computation of the dynamic variation of hemoglobin-3O2 saturation (SO2) within the vessels, irreversible sensitizer degradation due to photobleaching, and the microscopic distributions of 3O2, sensitizer concentration, and 1O2 dose deposition under various irradiation conditions. The simulations reveal severe axial gradients in 3O2 and in photodynamic dose deposition in response to a wide range of clinically relevant treatment parameters. Thus, unlike former Krogh cylinder-based models, which assume a constant 3O2 concentration at the vessel, this new model identifies conditions in which 3O2 depletion and minimal deposition of reacting 1O2 exist near the end of axial segments of vessels and shows that treatment-limiting 3O2 depletion is induced at fluence rates as low as 10 mW cm(-2). These calculations also demonstrate that intercapillary heterogeneity of photosensitizer contributes significantly to the distribution of photodynamic dose. This more rigorous mathematical model enables comparison with experimentally observable, volume-averaged quantities such as SO2 and the loss of sensitizer fluorescence through bleaching that have not been included in previous analyses. Further, it establishes some of the intrinsic limitations of such measurements. Specifically, our simulations demonstrate that tissue measurements of SO2 and of photobleaching are necessarily insensitive to microscopic heterogeneity of photodynamic dose deposition and are sensitive to intercapillary spacing. Because prior knowledge of intercapillary distances in tumors is generally unavailable, these measurements must be interpreted with caution. We anticipate that this model will make useful dosimetry predictions that should inform optimal treatment conditions and improve current clinical protocols.

Effects of Fluence Rate on Cell Survival and Photobleaching in Meta-Tetra-(hydroxyphenyl)chlorin–photosensitized Colo 26 Multicell Tumor Spheroids¶

Abstract We report the influence of fluence rate on the photobleaching and cell survival in Colo 26 multicell spheroids photosensitized by meta-tetra-(hydroxyphenyl)chlorin (mTHPC). Photosensitizer degradation and therapeutic efficacy increased dramatically and progressively when the fluence rate was reduced over the range from 90 to 5 mW cm−2. These experimental results were compared to a mathematical model of photobleaching based on self-sensitized singlet oxygen reactions with the photosensitizer ground state. This model incorporates photophysical parameters obtained from microelectrode measurements of oxygen depletion at the surface of mTHPC-sensitized spheroids and was refined by including the inhomogeneous distribution of mTHPC in spheroids and oxygen depletion in the bulk medium. Since the model is consistent with the experimental data we conclude that the fluence rate dependence of the cell survival and of mTHPC photobleaching is due to photochemical oxygen consumption and a predominantly singlet oxygen-mediated mechanism of mTHPC photobleaching. The threshold dose of reacting singlet oxygen was calculated to be 7.9 ± 2.2 mM in this system.

In Vivo mTHPC Photobleaching in Normal Rat Skin Exhibits Unique Irradiance-dependent Features¶

Abstract We report measurements performed on the normal skin of rats in vivo, which provide information on the photobleaching kinetics and mechanisms of the photosensitizer meso-tetrahydroxyphenyl chlorin (mTHPC). Loss of mTHPC fluorescence was monitored using in vivo fluorescence spectroscopy during photodynamic therapy (PDT) performed using 650 nm laser irradiation. The bleaching was evaluated for irradiances of 5, 20 and 50 mW cm−2. Two distinct phases of mTHPC photobleaching were observed. In the first phase there was no obvious irradiance dependence in the loss of fluorescence vs fluence. The second phase was initiated by an irradiance-dependent discontinuity in the slope of the bleaching curve, after which the photobleaching rates showed an irradiance dependence consistent with an oxygen-dependent reaction process. To investigate the unusual shape of the in vivo bleaching curves, we measured the PDT-induced changes in O2 concentrations in mTHPC-sensitized spheroids irradiated with 2, 5 and 20 mW cm−2 of 650 nm light. The oxygen concentration data indicated no unusual features within the range of fluences where the discontinuities in fluorescence were observed during in vivo spectroscopy. The fluorescence from the in vivo bleaching experiments thus reports a phenomenon that is not reported by measurements of the photochemical oxygen consumption in the spheroids.

Photochemical Oxygen Consumption Sensitized by a Porphyrin Phosphorescent Probe in Two Model Systems

Phosphorescence quenching of certain metalloporphyrins is used to measure tissue and microvascular pO(2). Oxygen quenching of metalloporphyrin triplet states creates singlet oxygen, which is highly reactive in biological systems, and these oxygen-consuming reactions are capable of perturbing tissue oxygenation. Kinetics of photochemical oxygen consumption were measured for a Pd-porphyrin in two model systems in vitro over a range of irradiances (1.34-134 mW cm(-2)). For a given irradiance, and, after correction for differing porphyrin concentrations, rates of oxygen consumption were similar when the Pd-porphyrin was bound to bovine serum albumin and when it was taken up by tumor cells in spheroids. At irradiances comparable to those used in imaging superficial anatomy, rates of oxygen consumption were sufficiently low (2.5 microM s(-1)) that tissue oxygenation would be reduced by a maximum of 6%. An irradiance of 20 mW cm(-2), however, initiated a rate of oxygen consumption capable of reducing tissue pO(2) by at least 20-40%. These measured rates of consumption impose limitations on the use of phosphorescence quenching in thick tissues. The irreversible photobleaching of the Pd-porphyrin was also measured indirectly. The bleaching branching ratio, 23 M(-1), is significantly lower than that of porphyrin photodynamic agents.

Activation of Heat Shock Protein 70 Promoter with meso‐Tetrahydroxyphenyl Chlorin Photodynamic Therapy Reported by Green Fluorescent Protein In Vitro and In Vivo¶

Cellular responses to photodynamic therapy (PDT) include induction of heat shock proteins (HSP). We examined meso‐tetrahydroxyphenyl chlorin (mTHPC) PDT–mediated HSP activation in EMT6 cells stably transfected with a plasmid containing the gene for green fluorescent protein (GFP) driven by an hsp70 promoter. mTHPC incubation induced concentration‐dependent GFP expression. Irradiation of cells exposed to a sensitizer concentration that induced a slight increase in GFP and no loss of cell viability resulted in fluence‐dependent GFP accumulation. In response to drug only and to PDT, GFP levels increased to a maximum of four‐ to five‐fold above control levels with increasing drug or fluence and then decreased at higher doses. A trypan blue–exclusion assay confirmed that decreased GFP levels in both cases were due to a loss of cell viability. For initial evaluation in vivo, HSP70/GFP–transfected EMT6 tumors were grown in BALB/c mice and subjected to mTHPC‐PDT with a fluence of 1 J/cm2. Six hours after PDT, GFP fluorescence was imaged in these tumors through the intact skin in vivo. These results indicate that sublethal doses of mTHPC‐PDT stimulate GFP expression under the control of an hsp70 promoter and illustrate the potential of noninvasively monitoring reporter protein fluorescence as a measure of molecular response to PDT.

ALA- and ALA-hexylester-induced protoporphyrin IX fluorescence and distribution in multicell tumour spheroids

Synthesis of protoporphyrin IX (PpIX) in intact murine mammary cancer cell spheroids is reported from optical sections obtained using a laser scanning confocal fluorescence microscope. EMT6 spheroids 275–350 μ m in diameter were incubated in 0.1–15 mM aminolevulinic acid (ALA) or 0.001–2 mM ALA-hexylester (h-ALA) to test the ability of both pro-drugs to diffuse into the spheroids and induce PpIX production. Spheroids incubated with ALA show significant fluorescence nonuniformity for all concentrations, with the outermost cells exhibiting greater porphyrin fluorescence. Comparable levels of fluorescence throughout the optical section are achieved with approximately 100-fold lower h-ALA concentrations, indicating that the interior cells maintain esterase activity and porphyrin synthesis and that h-ALA diffuses efficiently to the spheroid interior. Fluorescence gradients are less pronounced with h-ALA incubation, in part because of apparent saturation of esterase activity in the spheroid perimeter. Proliferating (Ki67 positive) and quiescent cell populations exhibit remarkably different h-ALA concentration dependencies. The incubation concentration resulting in maximum fluorescence with ALA is 10 mM, while the optimal concentration for h-ALA is 200-fold lower at 0.05 mM. Exceeding these optimal concentrations for both pro-drugs leads to an overall loss of fluorescence.