Mamta Khurana

Speckle variance detection of microvasculature using swept-source optical coherence tomography

We report on imaging of microcirculation by calculating the speckle variance of optical coherence tomography (OCT) structural images acquired using a Fourier domain mode-locked swept-wavelength laser. The algorithm calculates interframe speckle variance in two-dimensional and three-dimensional OCT data sets and shows little dependence to the Doppler angle ranging from 75 degrees to 90 degrees . We demonstrate in vivo detection of blood flow in vessels as small as 25 microm in diameter in a dorsal skinfold window chamber model with direct comparison with intravital fluorescence confocal microscopy. This technique can visualize vessel-size-dependent vascular shutdown and transient vascular occlusion during Visudyne photodynamic therapy and may provide opportunities for studying therapeutic effects of antivascular treatments without on exogenous contrast agent.

Simultaneous Two-photon Excitation of Photofrin in Relation to Photodynamic Therapy

Abstract Photodynamic therapy (PDT), the use of light-activated drugs (photosensitizers), is an emerging treatment modality for tumors as well as various nononcologic conditions. Single-photon (1-γ) PDT is limited by low specificity of the photosensitizer, leading to damage to healthy tissue adjacent to the diseased target tissue. One solution is to use simultaneous two-photon (2-γ) excitation with ultrafast pulses of near-IR light. Due to the nonlinear interaction mechanism, 2-γ excitation with a focused beam is localized in three dimensions, allowing treatment volumes on the order of femtoliters. We propose that this will be valuable in PDT of age-related macular degeneration (AMD), which causes blindness due to abnormal choroidal neovasculature and which is currently treated by 1-γ PDT. Here, Photofrin has been used as the photosensitizer to demonstrate proof-of-principle of 2-γ killing of vascular endothelial cells in vitro. The 2-γ absorption properties of Photofrin were investigated in the 750–900 nm excitation wavelength range. It was shown that 2-γ excitation dominates over 1-γ excitation above 800 nm. The 2-γ absorption spectrum of Photofrin in the 800–900 nm excitation wavelength range was measured. The 2-γ cross section decreased from about 10 GM (1 GM = 10−50 cm4 s/photon) at 800 nm to 5 GM at 900 nm. Adherent YPEN-1 endothelial cells were then incubated with Photofrin for 24 h and then treated by PDT at 850 nm where the 1-γ contribution was negligible. Cell death was monitored with the use of 2-γ scanning laser microscopy. The light doses required for killing were high (6300 J cm−2 for ∼50% killing), but 2-γ cytotoxicity was unequivocally demonstrated. Although Photofrin is, per se, not a good choice for 2-γ PDT due to its low 2-γ cross section, this work provides baseline data to guide the development of novel photosensitizers with much higher 2-γ cross sections (>100 GM), which will be required for 2-γ PDT of AMD (and other conditions) to be clinically practical.

Quantification of in vivo fluorescence decoupled from the effects of tissue optical properties using fiber-optic spectroscopy measurements

We present a method for tissue fluorescence quantification in situ using a handheld fiber optic probe that measures both the fluorescence and diffuse reflectance spectra. A simplified method to decouple the fluorescence spectrum from distorting effects of the tissue optical absorption and scattering is developed, with the objective of accurately quantifying the fluorescence in absolute units. The primary motivation is measurement of 5-aminolevulinic acid-induced protoporphyrin IX (ALA-PpIX) concentration in tissue during fluorescence-guided resection of malignant brain tumors. This technique is validated in phantoms and ex vivo mouse tissues, and tested in vivo in a rabbit brain tumor model using ALA-PpIX fluorescence contrast.

Quantitative In Vitro Demonstration of Two‐Photon Photodynamic Therapy Using Photofrin® and Visudyne®

Photodynamic therapy (PDT), the combined action of a photosensitizer and light to produce a cytotoxic effect, is an approved therapy for a number of diseases. At present, clinical PDT treatments involve one‐photon excitation of the photosensitizer. A major limitation is that damage may be caused to healthy tissues that have absorbed the drug and lie in the beam path. Two‐photon excitation may minimize this collateral damage, as the probability of absorption increases with the square of the light intensity, enabling spatial confinement of the photosensitizer activation. A potential application is the treatment of the wet‐form of age‐related macular degeneration, the foremost cause of central vision loss in the elderly.

One- and two-photon activated phototoxicity of conjugated porphyrin dimers with high two-photon absorption cross sections

Two-photon excited photodynamic therapy (PDT) has the potential to provide a highly targeted treatment for neoplastic diseases, as excitation can be pin-pointed to small volumes at the laser focus. In addition, two-photon PDT offers deeper penetration into mammalian tissue due to the longer wavelength of irradiation. Here we report the one-photon and two-photon excited PDT results for a collection of conjugated porphyrin dimers with high two-photon absorption cross sections. These dimers demonstrate high one-photon PDT efficacy against a human ovarian adenocarcinoma cell line (SK-OV-3) and exhibit no significant dark-toxicity at concentrations of up to 20 microM. Their one-photon excited PDT efficiencies, following irradiation at 657 nm, approach that of Visudyne, a drug used clinically for PDT. We investigated and optimised the effect of the photosensitizer concentration, incubation time and the light dose on the PDT efficacy of these dimers. These studies led to the selection of P2C2-NMeI as the most effective porphyrin dimer. We have demonstrated that P2C2-NMeI undergoes a two-photon activated process following excitation at 920 nm (3.6-6.8 mW, 300 fs, 90 MHz) and compared it to Visudyne. We conclude that the in vitro two-photon PDT efficacy of P2C2-NMeI is about twice that of Visudyne. This result highlights the potential of this series of porphyrin dimers for two-photon PDT.

Intravital high-resolution optical imaging of individual vessel response to photodynamic treatment

Intravital imaging using confocal microscopy facilitates high-resolution studies of cellular and molecular events in vivo. We use this, complemented by Doppler optical coherence tomography (OCT), to assess blood flow in a mouse dorsal skin-fold window chamber model to image the response of individual blood vessels to localized photodynamic therapy (PDT). Specific fluorescent cell markers were used to assess the effect on the vascular endothelial cell lining of the treated vessels. A fluorescently tagged antibody against an endothelial transmembrane glycoprotein (CD31) was used to image endothelial cell integrity in the targeted blood vessel. A cell permeability (viability) indicator, SYTOX Orange, was also used to further assess damage to endothelial cells. A fluorescently labeled anti-CD41 antibody that binds to platelets was used to confirm platelet aggregation in the treated vessel. These optical techniques enable dynamic assessment of responses to PDT in vivo, at both the vascular endothelial cell and whole vessel levels.

Drug and light dose responses to focal photodynamic therapy of single blood vessels in vivo

As part of an ongoing program to develop two-photon (2-gamma) photodynamic therapy (PDT) for treatment of wet-form age-related macular degeneration (AMD) and other vascular pathologies, we have evaluated the reciprocity of drug-light doses in focal-PDT. We targeted individual arteries in a murine window chamber model, using primarily the clinical photosensitizer Visudyne/liposomal-verteporfin. Shortly after administration of the photosensitizer, a small region including an arteriole was selected and irradiated with varying light doses. Targeted and nearby vessels were observed for a maximum of 17 to 25 h to assess vascular shutdown, tapering, and dye leakage/occlusion. For a given end-point metric, there was reciprocity between the drug and light doses, i.e., the response correlated with the drug-light product (DLP). These results provide the first quantification of photosensitizer and light dose relationships for localized irradiation of a single blood vessel and are compared to the DLP required for vessel closure between 1-gamma and 2-gamma activation, between focal and broad-beam irradiation, and between verteporfin and a porphyrin dimer with high 2-gamma cross section. Demonstration of reciprocity over a wide range of DLP is important for further development of focal PDT treatments, such as the targeting of feeder vessels in 2-gamma PDT of AMD.

Biodistribution and Pharmacokinetic Studies of a Porphyrin Dimer Photosensitizer (Oxdime) by Fluorescence Imaging and Spectroscopy in Mice Bearing Xenograft Tumors

Herein, we present a study of the pharmacokinetics and biodistribution of a butadiyne‐linked conjugated porphyrin dimer (Oxdime) designed to have high near‐infrared (NIR) 2‐photon absorption cross‐section for photodynamic therapy (PDT). Changes in biodistribution over time were monitored in mice carrying B16‐F10 melanoma xenografts, following intravenous injection. Using fluorescence imaging of live animals and analyzing isolated organs ex vivo at different time points between 30 min and 24 h after injection, accumulation of Oxdime was measured in several organs (heart, kidney and liver) and in tumor. The concentration in the plasma was about 5–10 times higher than in other tissues. The fluorescence signal peaked at 3–12 h after injection in most tissues, including the tumor and the plasma. The change in the fluorescence emission spectrum of the sensitizer over time was also monitored and a shift in the maximum from 800 to 740 nm was observed over 24 h, showing that the Oxdime is metabolized. Significant quantities accumulated in the tumor, indicating that this PDT sensitizer may be promising for cancer treatment.

Multi-Modality Optical Imaging of Vascular Responses to Photodynamic Therapy in Mouse Window Chamber Model

We demonstrate multi-modal optical imaging in a window-chamber vascular model to investigate the response to photodynamic therapy using novel photosensitizers with high 2-γ cross-section, and show that (micro)vessel closure is feasible at clinically-realistic doses.

Two-photon photodynamic therapy and its potential application to age related macular degenerations

Photodynamic therapy (PDT) using verteporfin is widely used for treatment of age related macular degeneration (AMD). Due to non-perfect selectivity of the drug accumulation in the neovasculature some collateral damage to healthy tissue arises during the treatment. Damage to healthy structures in the eye is always a concern because of a high probability of reducing visual acuity. Two-photon (2-&ggr;) photodynamic therapy potentially offers much higher treatment selectivity than its one-photon (1-&ggr;) counterpart. By utilizing focused light for 2-&ggr; excitation, treatment volumes on the order of microliters can be achieved thus maximizing localized insult to abnormal blood vessels and sparing healthy tissue. We propose that 2-&ggr; photodynamic therapy will be valuable in the treatment of choroidal neovascularization secondary to age related macular degeneration as well as other conditions. To ascertain feasibility of 2-&ggr; photodynamic therapy we measured 2-&ggr; spectrum and cross sections of verteporfin (80 GM at 940 nm, 1 GM = 10-50 cm4s/photon), chlorin e6 (14 GM at 800 nm) and tetrasulfonated aluminum phthalocyanine (140 GM at 900 nm) and investigated their in vitro efficiency under 2-&ggr; excitation. Only verteporfin demonstrated cell kill under the used irradiation parameters (average light intensity 9.1 mW, wavelength 850 nm, total light dose 6900 J/cm2). Dorsal skinfold window chamber model in mouse was used to test efficiency of 2-&ggr; PDT with verteporfin in vivo. Although we were able to induce photodynamic damage to a blood vessel using 1-&ggr; excitation, 2-&ggr; excitation resulted in no visible damage to irradiated blood vessel. The most probable reason is low efficiency of verteporfin as a 2-&ggr; photosensitizer. We also report 2-&ggr; spectrum of new photosensitizer, HCC4 (4300 GM at 830 nm), specifically designed for efficient 2-&ggr; excitation.