Aijun Gong

New Two-Photon Activated Photodynamic Therapy Sensitizers Induce Xenograft Tumor Regressions after Near-IR Laser Treatment through the Body of the Host Mouse

Purpose: The aim of this study was to show that novel photodynamic therapy (PDT) sensitizers can be activated by two-photon absorption in the near-IR region of the spectrum and to show, for the first time, that such activation can lead to tumor regressions at significant tissue depth. These experiments also evaluated effects of high-energy femtosecond pulsed laser irradiation on normal tissues and characterized the response of xenograft tumors to our PDT protocols. Experimental Design: Human small cell lung cancer (NCI-H69), non-small cell lung cancer (A549), and breast cancer (MDA-MB-231) xenografts were induced in SCID mice. Irradiation of sensitized tumors was undertaken through the bodies of tumor-bearing mice to give a treatment depth of 2 cm. Posttreatment tumor regressions and histopathology were carried out to determine the nature of the response to these new PDT agents. Microarray expression profiles were conducted to assess the similarity of responses to single and two-photon activated PDT. Results: Regressions of all tumor types tested were seen. Histopathology was consistent with known PDT effects, and no, or minimal, changes were noted in irradiated normal tissues. Cluster analysis of microarray expression profiling showed reproducible changes in transcripts associated with apoptosis, stress, oxygen transport, and gene regulation. Conclusions: These new PDT sensitizers can be used at a depth of 2 cm to produce excellent xenograft regressions. The tumor response was consistent with known responses to single-photon activated PDT. Experiments in larger animals are warranted to determine the maximal achievable depth of treatment.

Synthesis, characterization, and preclinical studies of two-photon-activated targeted PDT therapeutic triads

Photodynamic therapy (PDT) continues to evolve into a mature clinical treatment of a variety of cancer types as well as age-related macular degeneration of the eye. However, there are still aspects of PDT that need to be improved in order for greater clinical acceptance. While a number of new PDT photo-sensitizers, sometimes referred to as second- or third- generation therapeutic agents, are currently under clinical investigation, the direct treatment through the skin of subcutaneous tumors deeper than 5 mm remains problematic. Currently approved PDT porphyrin photo-sensitizers, as well as several modified porphyrins (e.g. chlorins, bacteriochlorins, etc.) that are under clinical investigation can be activated at 630-730 nm, but none above 800 nm. It would be highly desirable if new PDT paradigms could be developed that would allow photo-activation deep in the tissue transparency window in the Near-infrared (NIR) above 800 nm to reduce scattering and absorption phenomena that reduce deep tissue PDT efficacy. Rasiris and MPA Technologies have developed new porphyrins that have greatly enhanced two-photon absorption ( P A ) cross-sections and can be activated deep in the NIR (ca. 780-850 nm). These porphyrins can be incorporated into a therapeutic triad that also employs an small molecule targeting agent that directs the triad to over-expressed tumor receptor sites, and a NIR onephoton imaging agent that allows tracking the delivery of the triad to the tumor site, as well as clearance of excess triad from healthy tissue prior to the start of PDT treatment. We are currently using these new triads in efficacy studies with a breast cancer cell line that has been transfected with luciferase genes that allow implanted tumor growth and post- PDT treatment efficacy studies in SCID mouse models by following the rise and decay of the bioluminescence signal. We have also designed highly absorbing and scattering collagen breast cancer phantoms in which we have demonstrated dramatic cell kill to a depth of at least 4 cm. We have also demonstrated that at the wavelength and laser fluences used in the treatment of implanted tumors in the mouse mammary fat pads, there is little, if any, damage to the skin or internal mouse organs. In addition, we have also demonstrated that the implanted tumors can be treated to a depth of more than 1 cm by direct radiation through the dorsal side of the mouse.

Targeted two-photon photodynamic therapy for the treatment of subcutaneous tumors

Photodynamic therapy (PDT) has developed into a mature technology over the past several years, and is currently being exploited for the treatment of a variety of cancerous tumors, and more recently for age-related wet macular degeneration of the eye. However, there are still some unresolved problems with PDT that are retarding a more general acceptance in clinical settings, and thus, for the most part, the treatment of most cancerous rumors still involves some combination of invasive surgery, chemotherapy and radiation treatment, particularly subcutaneous tumors. Currently approved PDT agents are activated in the Visible portion of the spectrum below 700 nm, Laser light in this spectral region cannot penetrate the skin more than a few millimeters, and it would be more desirable if PDT could be initiated deep in the Near-infrared (NIR) in the tissue transparency window (700-1000 nm). MPA Technologies, Inc. and Rasiris, Inc. have been co-developing new porphyrin PDT designed to have greatly enhanced intrinsic two-photon cross-sections (>800 GM units) whose two-photon absorption maxima lie deep in the tissue transparency window (ca. 780-850 nm), and have solubility characteristics that would allow for direct IV injection into animal models. Classical PDT also suffers from the lengthy time necessary for accumulation at the tumor site, a relative lack of discrimination between healthy and diseased tissue, particularly at the tumor margins, and difficulty in clearing from the system in a reasonable amount of time post-PDT. We have recently discovered a new design paradigm for the delivery of our two-photon activated PDT agents by incorporating the porphyrins into a triad ensemble that includes a small molecule targeting agent that directs the triad to over-expressed tumor receptor sites, and a NIR one-photon imaging agent that allows the tracking of the triad in terms of accumulation and clearance rates. We are currently using these new two-photon PDT triads in efficacy studies with two breast cancer cell lines, both in vitro and in vivo. Both of these cell lines have been transfected with luciferase genes that allow implanted tumor growth and PDT efficacy to be monitored in living mouse models over time by following the rise and decay of the bioluminescence signals.

Synthesis, characterization and two-photon PDT efficacy studies of triads incorporating tumor targeting and imaging components

Over the past three years we have described the rationale for using new photosensitizers (PS) with greatly enhanced multi-photon absorption. In particular, we have synthesized new porphyrin-based photosensitizers that also incorporate small molecule targeting agents that direct the ensemble to over-expressed tumor receptor sites, as well as Near-infrared imaging agents that will allow practical image-guided two-photon PDT in the tissue transparency window (750-1000 nm) at laser fluences that are harmless to surrounding healthy tissue. We have previously shown (PW2006) successful treatment of human breast cancer models (MDA-MB-231) in SCID mice, and have recently extended these studies to the treatment of both human small cell (SC) (NCI-H69) and non-small cell (NSC) (A-459) models in SCID mice. We have demonstrated that lung cancer xenografts can be successfully treated by irradiating from the side of the mouse opposite the implanted tumor, thereby passing through ca. 2 cm of mouse skin, tissue and organs before encountering the bulk tumor. These results suggest that this technology can be used to treat deep subcutaneous spontaneous tumors in larger animal models (e.g. canine). We would also emphasize that the synthetic route to these triads attaches the targeting moiety in the last step of the synthesis, and can be easily changed, thus allowing a myriad of targeting agents to be employed, opening the door to the possibility of patient-specific PDT.

Broad bandwidth near-IR two-photon absorption in conjugated porphyrin-core dendrimers

We study two-photon absorption (2PA) spectra and absolute 2PA cross sections, as well as fluorescence emission- and excitation spectra along with time resolved fluorescence in a series of new dendrimers, where a single porphyrin/phthalocyanine core is decorated with electron-donating/two-photon absorbing groups. We show that if the combined system has strong π-conjugation/multipolar charge transfer between the core and the attached group(s), then the observed 2PA undergoes cooperative enhancement, where the peak 2PA cross section reaches σ2=600-2000 GM in a broad transition wavelength region 375-500 nm (laser wavelengths 750-1000 nm). In the systems with less conjugation, the 2PA is less enhanced, and in the limit of very weak conjugation maximum σ2 remains essentially the sum of the cross sections of the constituents. We show that the conjugation strength correlates with the fluorescence emission properties. In particular, in the strongly linked systems the fluorescence originates mostly from the core porphyrin, whereas in the weakly linked systems the attached chromophores emit independently from the porphyrin. In intermediate conjugation strength case we observe non-exponential fluorescence decays and fluorescence rise times, which indicated Forster resonant energy transfer from the side groups to the core.

Nanophotonic ensembles for targeted multi-photon photodynamic therapy

There has been a dramatic increase in the application of new technologies for the treatment of cancerous tumors over the past decade, but for the most part, the treatment of most tumors still involves some combination of invasive surgery, chemotherapy and radiation treatments. Photodynamic therapy (PDT), which involves the activation of an administered compound with laser light followed by a series of events leading to programmed cell death of the tumor, has been proposed as a noninvasive alternative treatment to replace the standard surgery/chemotherapy/radiation protocol. However, currently approved PDT agents operate in the Visible portion of the spectrum, and laser light in this region cannot penetrate the skin more than a few millimeters. Two-photon irradiation using more highly penetrating Near-infrared (NIR) light in the tissue transparency window (700-1000 nm) has been proposed for the treatment of subcutaneous tumors, but most porphyrins exhibit extremely small two-photon cross-sections. Classical PDT also suffers from the lengthy time necessary for accumulation at the tumor site, a relative lack of discrimination between healthy and diseased tissue, particularly at the tumor margins, and difficulty in clearing from the system in a reasonable amount of time. We have recently discovered a new design paradigm for porphyrins with greatly enhanced two-photon cross-sections, and are now proposing a nano-ensemble that would also incorporate small molecule targeting agents, and possibly one-photon NIR imaging agents along with these porphyrins in one therapeutic agent. Thus these ensembles would incorporate targeting/imaging/PDT functions in one therapeutic agent, and hold the promise of single-session outpatient treatment of a large variety of subcutaneous tumors.

Histopathological and expression profiling studies of early tumor responses to near-infrared PDT treatment in SCID mice

A novel class of porphyrin-based near-infrared photodynamic therapy (PDT) sensitizers is studied. We achieve regressions of human small cell lung cancer (NCI-H69), non-small cell lung cancer (A 459) and breast cancer (MDAMB- 231) xenografts in SCID mice at significant tissue depth by irradiation with an amplified femtosecond pulsed laser at 800 nm wavelength. Significant tumor regressions were observed during the first 10-14 days post treatment. Tumor histopathology was consistent with known PDT effects, while no significant changes were noted in irradiated normal tissues. In vivo imaging studies using intravenous injections of fluorescent dextran demonstrated an early loss of tumor blood flow. RNA was isolated from NCI-H69 PDT treated SCID mouse xenografts and paired untreated xenografts at 4 hours post laser irradiation. Similarly RNA was isolated from PDT treated and untreated Lewis lung carcinomas growing in C57/Bl6 mice. Expression profiling was carried out using AffymetrixTM human and mouse GeneChips®. Cluster analysis of microarray expression profiling results demonstrated reproducible increases in transcripts associated with apoptosis, stress, oxygen transport and gene regulation in the PDT treated NCI-H69 samples. In addition, PDT treated Lewis lung carcinomas showed reproducible increases in transcripts associated with immune response and lipid biosynthesis. PDT treated C57/Bl6 mice developed cytotoxic T cell activity towards this tumor, while untreated tumor bearing mice failed to do so.

New two-photon materials for fast volumetric rewritable optical storage

Two-photon absorption (TPA) is a promising technique for high density 3D-addressing for writing and read-out of data, provided that suitable two-photon sensitive materials facilitating fast recording and read-out will be developed. Free-base porphyrins and other metal-free tetrapyrroles, such as phthalocyanies and naphthalocyanines possess a unique fast intrinsic photo-tautomerization mechanism, which consists in switching the position of a pair of protons in the core of the molecule. In the past photo-tautomerization was used for holographic storage, but can be also applied for bit-oriented volumetric information storage using laser-excited fluorescence for readout. However, the utility of the photo-tautomerization for two-photon storage was severely restricted so far by the fact that all known tetrapyrroles have rather low TPA cross section, with values not exceeding 1 - 10 GM (1GM = 10-50 cm4 s photon-1). Recently we have discovered a new class of porphyrins, where TPA cross section is dramatically amplified by certain chemical modification of the chemical structure, and that some of the new porphyrins have the ability of photo-tautomerization by simultaneous absorption of two photons. In this paper we discuss the photophysics and nonlinear optics of the new porphyrins that can lead to fast volumetric re-writable optical storage. We present a quantitative comparison of the new compounds with previously known TPA chromophores and introduce a merit figure, which takes into account both TPA cross-section as well as the efficiency of light-induced changes. We show the combination of high cross sections of two-photon absorption, up to 1000 GM in near-IR range of wavelength, with the fast photo-tautomerization, offers, for the first time, a sufficiently high merit figure needed for implementation of high-density, high speed volumetric two-photon re-writable optical storage.