In vivo PDT dosimetry: singlet oxygen emission and photosensitizer fluorescence

In vivo PDT dosimetry: singlet oxygen emission and photosensitizer fluorescence Seonkyung Lee* a, Kristin L. Galbally-Kinney a, Brian A. Murphy a, Steven J. Davis a, Tayyaba Hasan b, Bryan Spring b, Yupeng Tu, Brian W. Pogue c, Martin E. Isabelle c, and Julia A. O Hara c a Physical Sciences Inc., 20 New England Business Center, Andover, MA, USA 01810-1077 b Massachusetts General Hospital, Wellman Laboratories of Photomedicine, 40 Blossom Street, Boston, MA, USA 02114-2605 c Dartmouth-Hitchcock Medical Center, One Medical Center Drive, Lebanon, NH 03756 ABSTRACT Photodynamic therapy (PDT) is a light activated chemotherapy that is dependent on three parameters: photosensitizer (PS) concentration; oxygen concentration; and light dosage. Due to highly variable treatment response, the development of an accurate dosimeter to optimize PDT treatment outcome is an important requirement for practical applications. Singlet oxygen is an active species in PDT, and we are developing two instruments, an ultra-sensitive singlet oxygen point sensor and a 2D imager, with the goal of a real-time dosimeter for PDT researchers. The 2D imaging system can visualize spatial maps of both the singlet oxygen production and the location of the PS in a tumor during PDT. We have detected the production of singlet oxygen during PDT treatments with both in-vitro and in-vivo studies. Effects of photobleaching have also been observed. These results are promising for the development of the sensor as a real-time dosimeter for PDT which would be a valuable tool for PDT research and could lead to more effective treatment outcome. We summarize recent results in this paper. Keywords: Photodynamic therapy, singlet molecular oxygen, PDT dosimetry 1. INTRODUCTION Photodynamic therapy (PDT) continues to advance and to show promise as a targeted therapy for some cancers. PDT uses certain compounds known as photosensitizers (PSs) that are preferentially retained in malignant tumors. Stimulated with visible light, the photosensitizers initiate a reaction that selectively kills the malignant cells to which they are attached. This photo-induced process uses an excitation source, typically a low power laser, to produce the first excited state of molecular oxygen, (O 2 (a 1 Δ g )) also known as singlet molecular oxygen, that has known cytotoxic effects on cancer cells 1-3. The singlet O 2 destroys the cancer cells via two distinct mechanisms 2,3 : a) direct disruption of cell function by rupturing of the cell wall or destruction of the cell mitochondria, and b) constriction of the vascular network in the tumor that provides nutrients to the tumor. The targeted nature of PDT derives from its preferential retention in tumors. Consequently, there has been considerable interest in developing a sensor for singlet oxygen produced by the PDT process. Correlations of the singlet O 2 produced with treatment efficacy could be one important use of such a sensor. Some researchers have attempted to develop dosimeters based on the fluorescence intensity of the PS in the tumor, but photobleaching of the PS precludes this as an accurate method. We and others have been attempting to develop sensitive monitors for singlet O 2 for use in PDT 4-11. Recently 5,6 we described initial in-vivo results demonstrating singlet O 2 production in a tumor model in rats and in the skin of healthy human subjects. In this paper we describe improvements to our sensors and discuss in-vitro studies designed to demonstrate high sensitivity detection of singlet oxygen prior to planned further in-vivo experiments. *lee@psicorp.com; phone 1 978 689-0003; fax 1 978 689-3232; psicorp.com

The fundamental type II PDT process is illustrated in Figure 1. S 1 Laser Excitation S 0 Triplet States Singlet States T 2 Intramolecular Energy Transfer Collisional (t = 10-8 Transfer to Oxygen s) K (S -T1 ) K (T 1-1 O 2 ) 1 T 1 A S 1 K Q S 1 Emission Quenching Visible Fluorescence A T 1 Emission K 1 Quenching 1.27 m Emission O 2 ( 1 ) O 2 ( 3 ) Photosensitizer Oxygen B-3271c Figure 1. Diagram of the type II PDT process. In general, the photosensitizer absorbs light, which excites it to the first excited singlet state. The excited singlet state strongly radiates to the ground singlet state emitting optical radiation characteristic of the PS. Typically, this contains a visible component that can be used to locate the tumor and its boundaries and is appropriate for imaging studies using Si-based CCD arrays. The excited singlet state of the PS also has a large probability of intrasystem crossing to a metastable triplet state as shown above. In many photosensitizers this triplet state is nearly resonant with the transition of oxygen from ground state to excited singlet state. Collisions between this metastable PS molecule and ground state oxygen (present in the tumor) populate the a 1 Δ g state via an energy transfer process. The singlet O 2 emits very weakly in the near infrared near 1.27 μm, and this emission forms the basis of our current singlet oxygen detection systems. Prompt dye fluorescence from the 1 S 0 state has a lifetime on the order of 10 ns since it is from a radiatively allowed transition. It decays much more rapidly than the emissions from the singlet O 2 (lifetime of 4 µs in aqueous media and as short as 0.1 s in biological media). The observed lifetime of singlet O 2 varies greatly depending on its environment. For example, in table 1 we show results from some of our previous work 4 where we produced singlet O 2 via the PDT process in several liquids including: acetone, methanol, and water. Table 1. Summary of lifetimes measured in several media PS C -e6 T4SP A P c S4 Solvent Acetone Methanol Water Water with 5% FBS Methanol Water Methanol Water O 2 ( 1 ) Lifetime ( s) 49.5 9.4 3.7 0.4 9.7 4.4 10.6 4.3 Dye Triplet State Lifetime T ( s) 0.4 0.5 0.9 5.0 0.7 2.7 0.6 2.0

We use fiber coupled, pulsed diode lasers as our excitation sources and can tailor the excitation wavelength to optimize coupling into the particular PS being studied. We observe the singlet O 2 emission subsequent to the termination of the diode laser pulse in order to minimize interference from the prompt and much more intense PS fluorescence that is present even at 1.27 µm while the diode laser is on. Indeed, our system can monitor both the near IR tail of the PS fluorescence and the singlet O 2 emission at 1.27 μm using a near-ir photomultiplier tube (PMT) with a time response < 5 ns. This PMT has low enough dark current that we can use photon counting methods to optimize the sensitivity. 2. EXPERIMENTS AND RESULTS In Figure 2 we illustrate typical signals recorded with our near-ir PMT-based point sensor. Figures 2a and b show data for the PS C -e6 in acetone (Figure 2a) and water (Figure 2b) for a 5 s diode laser excitation pulse width. The temporal evolution of the production of singlet O 2 (via transfer from the PS triplet state) and its subsequent quenching by the solvent molecules are evident in these data. For the acetone solution (Figure 2a), the quenching is relatively weak and the singlet O 2 emission by the end of the diode laser pulse is several times stronger than the near-ir fluorescence from the PS. In contrast, for the more severe quenching aqueous environment, the singlet O 2 emission is much weaker. The dramatic reduction in due to water quenching when compared to acetone, a relatively weak quencher of singlet O 2. Note in both media, while the diode laser is on there is a growth in the observed signal due to the production of singlet O 2 during the flat-top laser pulse. Thus, we can observe both the production of singlet O 2 while the laser is on and the evolution of the emission subsequent to the laser pulse. Relative Intensity 400 350 300 250 200 150 100 50 Singlet Oxygen Emission Increasing Laser Off Laser On O 2 ( 1 ) Luminescence 0 0 20 40 60 Time (µs) (a) Relative Intensity 160 140 120 100 80 60 40 20 O 2 ( 1 ) Luminescence and Dye Fluorescence Laser Off O 2 ( 1 ) Luminescence. (Dye Fluorescence Has DecayedtoZero) 0 0 20 40 60 Time (µs) (b) F-7185 Figure 2. Temporal evolution of the IR emission centered at 1.27 m following 5 s excitation of C -e6. (a) acetone, (b) water. The temporal evolution shown in Figure 2b is typical of the singlet O 2 signatures that we observe even in-vivo. Most of the bright PS fluorescence promptly terminates at the end of the diode laser pulse, and we sum the intensity (photoelectron counts) after the diode laser is shut off to obtain our singlet O 2 signal. However, in tissue, the singlet O 2 becomes so highly quenched that some weak emitters can cause spectral interferences, even when observing during the time that the diode laser is off. The relatively slow emission (phosphorescence) from triplet state of the PS is a potential interference for in-vivo studies. The triplet state lifetime is typically on the order of microseconds and the emission (albeit weak) can occur subsequent to the diode laser pulse. This requires additional optical filtering to isolate the singlet O 2 spectral feature from the broadband emission from the PS. We use a series of three narrow band interference filters with center wavelengths of 1.22, 1.27, and 1.32 μm to spectrally discriminate between the PS and singlet O 2 emission. This provides a means for subtracting background emission not specifically due to singlet O 2 that emits in a narrow (18nm) band centered at 1.27µm. With this approach we have succeeded in detecting singlet O 2 production from two photosensitizers in tumors implanted in rats and from healthy human skin containing topical ALA photosensitizer. 5,6

However, the signal to noise ratios of the signals from our initial in-vivo studies are much weaker than that shown in Figure 2. Subsequently, we have improved the sensitivity of our detection system and its discrimination of the singlet O 2 emission. Recall that we use three narrow bandpass optical filters to examine the emission at 1.22, 1.27, and 1.32µm. Figure 3 shows the temporal evolution of these three wavelengths during and subsequent to the diode laser excitation pulse in aqueous solution with triton X-100 (TTX-100). We also observed this behavior with fetal bovine serum (FBS) solution, protein laden solution, that is a more severe quenching environment than water, and is more representative of what will be encountered in tissue. For these data we used the PS verteporfin (BPD-MA) at a 25 µm concentration. The emission at 1.27µm indicates a slow growth of emission during the diode laser pulse and a continued growth followed by a diminution subsequent to the termination of the diode laser pulse. These are the raw data prior to subtraction of the background signal. Figure 3. Temporal evolution of the observed emission signals at 1.22, 1.27, and 1.32µm when irradiating an aqueous BPD with TTX-100. Figure 4 shows the background subtracted emission at 1.27µm due to the singlet O 2 emission subsequent to the termination of the diode laser pulse that occurred at 11μs on the time scale of Figure 4. The increase of the singlet O 2 signal immediately after the diode laser pulse is evident prior to the subsequent quenching of the singlet O 2 signal. This behavior is similar to that shown in Figure 2b for a water solution with much less quenching. The increased noise is due to the larger photoelectron noise values during the diode laser pulse. Nevertheless, we are able to observe the entire temporal evolution of the singlet O 2 during and after the diode laser pulse. This represents a significant enhancement of the sensitivity of our sensor.

20 15 10 5 0-5 5 10 15 20 25 30 Time (µs) K-2515 Figure 4. Background subtracted singlet oxygen emission subsequent to the termination of the diode laser pulse for an aqueous solution of BPD with TTX-100. The imaging version of our singlet oxygen sensor uses a sensitive InGaAs camera to detect both the PS fluorescence and the singlet O 2 emission. Similar to the point sensor described above, we use three optical filters to spectrally isolate the singlet O 2 emission in the 1.27μm spectral region. Figure 5 illustrates the major components of the imaging sensor. Note that a second, visible wavelength CCD camera is used to simultaneously monitor the strong visible fluorescence from the PS. IR Camera Diode Laser Filter Slider Beam Splitter L Filter Computer Diode Laser Power Supply L L VIS Camera TC Camera Controller J-4649 Tumor Figure 5. Schematic of the imaging sensor. A configuration appropriate for in-vivo studies is shown. We have completed a systematic characterization of this system including extensive studies of the dependence of the singlet O 2 signal as a function of PS in FBS solutions. Figure 6 shows images of spatially resolved singlet O 2 emission and BPD fluorescence in PBS solutions. For these data, the fiber coupled diode laser beam was directed into a cuvet, and the two cameras imaged the irradiation area. The left panel shows images of the singlet oxygen and PS species in an oxygenated solution. The right panel shows data after the solution has been purged with nitrogen gas to remove the oxygen. The singlet O 2 is absent when the oxygen is removed, confirming that oxygen is required in the solution in order to observe the singlet O 2 emission signal. In contrast, the PS signal is independent of the dissolved oxygen in the solution.

Figure 6. Images of singlet oxygen emission and BPD fluorescence in PBS solutions. The concentration of BPD for these data was 25 µm. Currently, we are completing a series of in-vitro studies to determine the ultimate sensitivity of the imaging system in preparation for a limited animal study using a pancreatic tumor model. 3. SUMMARY We have described two sensors that we are developing as dosimeters for PDT. The non-imaging PMT-based system is more sensitive, but the imaging system appears to have sufficient sensitivity to observe singlet oxygen emission in-vivo. We are continuing to improve the sensitivity and are quantifying the absolute detection limits of singlet oxygen. We plan to complete in-vivo studies in the near future with the goal of demonstrating dosimetric measurements that are relevant to PDT treatments. 4. AKNOWLEDGEMENTS This work was supported by the National Cancer Institute under SBIR grants 4R44C128364-02 and 2R44CA119486-02. We are very grateful for this support. REFERENCES [1] Wieshaupt, K.R., Gomer, C.J. and Dougherty, T.J., Identification of Singlet Oxygen as the Cytoxic Agent in Photo-inactivation of a Murine Tumor, Cancer Res. 36, 2326 (1976). [2] Kessel, D., Castelli, M. and Reiners, J., On the Mechanism of PDT-induced Mitochondrial Photodamage, Proc. SPIE 4248, 157 (2001). [3] Fingar, V.H., Wieman, T.J., Wichle, S.A. and Cerrito, P.B., The Role of Microvascular Damage in Photodynamic Therapy, The Effect of Treatment on Vessel Constriction, Permeability, and Leukocyte Adhesion, Cancer Research 52, 4914-4921 (1992). [4] Davis, S.J., Zhu, L., Minhaj, A.M., Hinds, M.F., Lee, S., Keating, P.B., Rosen, D.I. and Hasan, T., Ultra-sensitive, Diode Laserbased Monitor for Singlet Oxygen, Proc. SPIE, 4952, (2003).

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