Photodynamic nanoplatforms offer alternative to standard cancer treatments
Raoul Kopelman and Rodney R. Agayan
New photodynamic cancer therapies combine the tumor detection and imaging capabilities of nanoparticles with the increased penetration depth of infrared two-photon excitation.

Traditional photodynamic therapy (PDT) is based on photoactive drugs that produce excited singlet-state oxygen molecules from local (ground-state) oxygen molecules under illumination with visible light.1 The singlet oxygen produces reactive oxygen species, primarily free radicals such as ·OH. These free radicals attack DNA and other vital biomolecules in the cell, leading to cell death. Due to the limited tissue penetration of visible light, PDT has mostly been utilized for skin cancers.
Often injected into the area of the tumor, the photodynamic drug is composed of non-toxic molecules that efficiently absorb red wavelengths. Excitation of these molecules, usually a triplet state excitation deriving its energy from the excited singlet state, is quenched by oxygen molecules through an energy exchange that excites the oxygen molecule from its ground triplet state to its excited singlet state. This “singlet oxygen” is often called “killer oxygen,” as it quickly reacts with neighboring molecules, including water, to produce highly-reactive free radicals that, in turn, kill the cell. The United States Food and Drug Administration (FDA)-approved PDT drug is made of red blood cell hemoglobin-based molecules (trade name Photofrin), but other PDT molecules are awaiting FDA approval, and several are approved in Canada and Europe.
PDT has three primary problems. First, PDT is limited to superficial tumors, or tumors that are reachable by optical fibers. Second, the PDT drug molecule is not very selective and thus kills neighboring healthy cells. Third, a lesser concern is the photo-toxicity of the drug; treated patients must be kept in the dark for days or longer. At the same time, PDT has several advantages: the therapeutic laser beam can be selectively aimed at the tumor only; the visible radiation (usually 488nm or 633 nm) will not penetrate far beyond the tumor, even if non-tumor cells contain the drug; and PDT will not cause damage to cells that neither the light nor the drug reach. Thus, compared to chemotherapy and radiation therapy, PDT is quite benign. The question that now arises is whether we can extend this approach to the treatment of non-superficial tumors or to tumors that are diagnosed non-visually (with later confirmation by biopsy).
Some researchers have suggested the use of infrared active dyes in PDT,2 as the activating infrared photons can penetrate deeper into tissue than visible light. An analogous approach involves pulsed infrared photons used in combination with two-photon-absorbing PDT drug molecules.3 While this method also provides deeper photon penetration, it exacerbates the non-selectivity of the method as well: more non-tumorous tissue will be harmed.
The new PDT paradigm addressed here consists of nanoplatforms, which are nanoparticles (NP) that combine PDT with tumor detection and tumor imaging (see Figure 1). These NP, when injected into the bloodstream, find the cancer cells and enter them using selective molecular targeting. At the same time, they enable external MRI or optical imaging of the tumor. This allows one to aim a laser, via an optical fiber, at the tumor.

Figure 1. Schematic of a photodynamic nanoplatform for cancer therapy.
The NP PDT approach has been demonstrated4 on rats bearing the deadliest human brain cancer, 9L Glioma. Control mice with 5mm brain tumors survive only one week; those treated with existing therapeutic approaches live less than two weeks. But 60% of NP PDT treated rats were cured of their brain tumors. The procedure involved only 5min illumination by a 1W helium-neon laser, using a fiber inserted through the skull with a hemispherical diffuser.
With the same tumor, survival time for humans is only 4–6 months, and neither chemotherapy nor radiation therapy are effective. More details on these nano-drugs, including their synthesis, composition, and toxicology, are given in a paper by Yong-Eun Koo et al.5
The latest additions to these “photonic drugs” are PDT NPs that utilize two-photon excitation.6 Using an experimental two-photon PDT dye (embedded in the NP) and a pulsed Ti:sapphire laser, we demonstrated the effectiveness of this technique on live breast-cancer cells (see Figures 2 and 3).

Figure 2. Demonstration of cell-kill induced by two-photon photodynamic excitation. Fluorescent cell stains indicate live cells (calcein, green) or dead cells (propidium iodide, red). (a-d) Rat C6 glioma cells exposed to a 100mW/cm2, 780nm laser for 1min. Images were taken at different times: (a) before irradiation; (b) immediately after irradiation; (c) 40min after irradiation; (d) 120min after irradiation. (e-h) Rat C6 glioma cells incubated with 1mg/mL two-photon photosensitizing dye-encapsulated nanoparticles and exposed to a 100mW/cm2, 780nm laser for 1min. Images were taken at different times: (e) before irradiation; (f) immediately after irradiation; (g) 30min after irradiation; (h) 130min after irradiation


Figure 3. Demonstration of cell-kill localization. Imaging the cells with 10×-reduced magnification after excitation reveals that only cells in close proximity to the two-photon excitation focus are affected. This is indicated by both red fluorescence and a change in shape of the cells. Image acquisition occurred 135min after irradiation.
Once more-efficient two-photon dye-based NP are perfected, a second generation of such nano-drugs should work not only for highly-transparent brain tumors but also for other tumors in areas of the body with lower optical transparency.

Raoul Kopelman
Department of Chemistry, Department of Physics, Department of Applied Physics,
University of Michigan
Ann Arbor, MI
Raoul Kopelman is the Richard Smalley Distinguished University Professor of Chemistry, Physics, and Applied Physics at the University of Michigan, Ann Arbor, as well as a member of the Biophysics Program, Biomedical Engineering, and the Center for Biological Nanotechnology in the Medical School. His current research interests are in non-classical chemical reaction kinetics and in ultra-small opto-chemical sensors and actuators for biomedical use.
Rodney R. Agayan
Department of Applied Physics, Department of Chemistry,
University of Michigan
Ann Arbor, MI 
Rodney R. Agayan received his BS in Applied and Engineering Physics in 1997 from Cornell University. He worked as a research scientist at the Lawrence Livermore National Laboratory in Livermore, CA, then received MS degrees in Applied Physics (2003) and Electrical Engineering (2005) at the University of Michigan. He is currently a PhD candidate in Applied Physics at the University of Michigan where his interests include laser tweezers and micro- and nanoparticle biosensors.

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DOI: 10.1117/2.1200702.0630

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