Nanotechnology has the potential to deliver multiple imaging and therapeutic agents to the "right place at the right time". This could dramatically improve treatment responses in cancer which
have been so far, dismal as well as allow us to monitor this response online. Pancreatic cancer (PanCa) has a poor prognosis with a 5-year survival rate of only 5% and there is a desperate need
for effective treatments. Photodynamic therapy (PDT) has shown promising results in treating PanCa. Mechanism-based combinations with PDT have enhanced treatment outcome. Agents
tested with PDT include Avastin, an antibody against vascular endothelial growth factor (VEGF) which is approved for treating various cancers. Here, we investigate the effect of neutralizing
intracellular VEGF using nanotechnology for the delivery of Avastin in combination with PDT.
For this we used a construct called "nanocells" in which the photosensitizer was trapped inside polymer nanoparticles and these, with Avastin, were then encapsulated inside liposomes.
Simultaneous delivery of drugs in nano-constructs could improve the treatment response of mechanism based combination therapies against cancer. Our studies demonstrate significant enhancement in treatment outcomes when nanocell-based PDT is combined with Avastin in
orthotopic PanCa mouse models. We propose a new paradigm for Avastin-based therapy by combining intracellular delivery of the antibody and PDT using nanotechnology for treating PanCa.
PDT is clinically useful for precancers (actinic keratoses; AK) of the skin, but the optimal duration for 5-ALA
application is still controversial. For basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), cure rates remain
inferior to surgical excision. Lack of knowledge about regional levels of PpIX levels within target tissues clearly
contribute to these suboptimal results. To investigate PpIX levels achievable in human skin neoplasias in-vivo, a clinical
study to monitor PpIX accumulation in vivo was performed. PpIX-fluorescence in patients undergoing ALA-PDT for
facial AK was monitored via real-time in-vivo fluorescence dosimetry, with measurements q20 min following
application of 5-ALA (Levulan Kerastick). PpIX accumulation followed linear kinetics in nearly all cases. The slopes
varied widely, and did not correlate with clinical outcome in all patients. Some patients with a low accumulation of
PpIX fluorescence had a good response to therapy, whereas others with high PpIX accumulation required repeat
treatment (although not necessarily of the same lesion). PpIX accumulation rates did correlate to a certain degree with
the overall amount of erythema. We conclude that unknown factors besides PpIX levels must be critical for the response
to treatment. To assess the relationship between PpIX levels in various skin cancers, patients undergoing routine Mohs
surgery for BCC or SCC were measured by in-vivo dosimetry at 2 h after 5-ALA application. Overall, a progressive
increase in PpIX signal during malignant progression was observed, in the following rank order: Normal skin < AK <
SCC ~ BCC.
Pancreatic cancer (PanCa) has a poor prognosis with a 5-year survival rate of only 5%. Photodynamic therapy (PDT) has
shown promising results in treating PanCa. Mechanism-based combinations with PDT have enhanced treatment
outcome. Agents tested with PDT include Avastin, an antibody against vascular endothelial growth factor (VEGF) which
is approved for treating various cancers. Simultaneous delivery of drugs in nano-constructs could improve the treatment
response of mechanism based combination therapies. Here, we investigate the effect of neutralizing VEGF using
nanotechnology for the delivery of Avastin in combination with PDT. For this we used a construct called "nanocells" in
which the photosensitizer was trapped inside polymer nanoparticles and these, with Avastin, were then encapsulated
inside liposomes. In vitro, nanocells containing Avastin (NCA) significantly enhanced cytotoxicity in PanCa cells. NCA
based PDT also significantly improved treatment response in mice that were orthotopically implanted with PanCa.
Avastin delivered extracellularly in combination with PDT did not show any improvement. Here we propose a new
paradigm for Avastin-based therapy by combining intracellular delivery of the antibody and PDT using nanotechnology
for treating PanCa.
Photodynamic therapy (PDT) is a viable treatment option for a wide range of applications, including oncology, dermatology, and ophthalmology. Singlet oxygen is believed to play a key role in the efficacy of PDT, and on-line monitoring of singlet oxygen during PDT could provide a methodology to establish and customize the treatment dose clinically. This work is the first report of monitoring singlet oxygen luminescence in vivo in human subjects during PDT, demonstrating the correlation of singlet oxygen levels during PDT with the post-PDT photobiological response.
Cytokines are important messengers in cell-to-cell communications that regulate vital cellular and physiological
processes, and play an important role in defining the diagnosis, prognosis and treatment response in various diseases.
Although current ex vivo biochemical assays for cytokine quantitation are useful, their capabilities for studying dynamic
cytokine expression in living systems are limited. Optical molecular imaging technology can help probe the
spatiotemporal dynamics of cytokine expression in vivo and in real-time. We developed an in vivo optical molecular
imaging strategy for monitoring one of these cytokines, the vascular endothelial growth factor (VEGF). With the
imaging strategy, changes in tumoral VEGF concentration following cobalt chloride treatment and photodynamic
therapy (PDT) were monitored. This was the first systematic study to test the feasibility of VEGF-targeted molecular
imaging, and can potentially set the basis for online monitoring of cytokines that will help develop effective tools for
diagnosis, prognosis, treatment planning and monitoring.
Recent advances in light sources, detectors and other optical imaging technologies coupled with the development of
novel optical contrast agents have enabled real-time, high resolution, in vivo monitoring of molecular targets. Noninvasive
monitoring of molecular targets can help optimize photodynamic therapy (PDT) by providing the capabilities to
monitor the efficacy of treatment. Our lab has developed optical imaging technologies to investigate a wide range of
molecular, physiological and morphological responses to photodynamic therapy (PDT). With the idea that drug delivery
to the different compartments in the tumor is an important determinant of the treatment effect, we studied drug delivery
in vitro and in vivo using optical imaging tools. A molecular specific contrast agent that targets the vascular endothelial
growth factor (VEGF) was developed to monitor the changes in the protein expression following PDT. We also studied
the PDT-induced physiological changes in vascular permeability and metastasis with in vivo imaging.
We present a mathematical model to calculate the relative concentration of light scatterers, light absorbers, and fluorophores in the epithelium and stroma. This mathematical description is iteratively fit to the fluorescence spectra measured in vivo, yielding relative concentrations of each molecule. The mathematical model is applied to a total of 493 fluorescence measurements of normal and dysplastic cervical tissue acquired in vivo from 292 patients. The estimated parameters are compared with histopathologic diagnosis to evaluate their diagnostic potential. The mathematical model is validated using fluorescence spectra simulated with known sets of optical parameters. Subsequent application of the mathematical model to in vivo fluorescence measurements from cervical tissue yields fits that accurately describe measured data. The optical parameters estimated from 493 fluorescence measurements show an increase in epithelial flavin adenine dinucleotide (FAD) fluorescence, a decrease in epithelial keratin fluorescence, an increase in epithelial light scattering, a decrease in stromal collagen fluorescence, and an increase in stromal hemoglobin light absorption in dysplastic tissue compared to normal tissue. These changes likely reflect an increase in the metabolic activity and loss of differentiation of epithelial dysplastic cells, and stromal angiogenesis associated with dysplasia. The model presented here provides a tool to analyze clinical fluorescence spectra yielding quantitative information about molecular changes related to dysplastic transformation.
Recent advances in light sources, detectors and other optical imaging technologies coupled with the development of novel optical contrast agents have enabled real-time, high resolution, in vivo monitoring of molecular targets. Noninvasive monitoring of molecular targets is particularly relevant to photodynamic therapy (PDT), including the delivery of photosensitizer in the treatment site and monitoring of molecular and physiological changes following treatment. Our lab has developed optical imaging technologies to investigate these various aspects of photodynamic therapy (PDT). We used a laser scanning confocal microscope to monitor the pharmacokinetics of various photosensitizers in in vitro as well as ex vivo samples, and developed an intravital fluorescence microscope to monitor photosensitizer delivery in vivo in small animals. A molecular specific contrast agent that targets the vascular endothelial growth factor (VEGF) was developed to monitor the changes in the protein expression following PDT. We were then able to study the physiological changes due to post-treatment VEGF upregulation by quantifying vascular permeability with in vivo imaging.
In the context of clinical trials, calibration protocols for optical instruments that ensure measurement accuracy and the ability to carry out meaningful comparisons of data acquired from multiple instruments are required. A series of calibration standards and procedures are presented to assess technical feasibility of optical devices for cervical precancer detection. Measurements of positive and negative standards, and tissue are made with two generations of research grade spectrometers. Calibration accuracy, ability of standards to correct and account for changes in experimental conditions, and device components are analyzed. The relative frequency of measured calibration standards is investigated retrospectively using statistical analysis of trends in instrument performance. Fluorescence measurements of standards and tissue made with completely different spectrometers show good agreement in intensity and lineshape. Frequency of wavelength calibration standards is increased to every 2 h to compensate for thermal drifts in grating mount. Variations in illumination energy detected between standards and patient measurements require probe redesign to allow for simultaneous acquisition of illumination power with every patient measurement. The use of frequent and well-characterized standards enables meaningful comparison of data from multiple devices and unambiguous interpretation of experiments among the biomedical optics community.
Optical excitation of chemicals provides reactive excited states, which can initiate chemical reactions and can also relax via radiative photophysical processes, providing the basis for fluorescence diagnostics. The best-known example of the former is Photodynamic Therapy (PDT), which is now approved for the treatment of a number of neoplastic and non-neoplastic pathologies. Although the concept of the use of photodynamic agents in diagnostics is as old as their use in therapy, the focused development of this aspect has been relatively recent. Typically, photodynamic agents have high triplet yields and relatively long triplet lifetimes (microsecond range), which allows them to interact and destroy molecular targets near them either directly or indirectly by producing other toxic molecular species. Associated with a high triplet yield is the fortunate attribute of most PDT agents in having low but finite fluorescence quantum yields. Fluorescence from these molecules may be used not only for diagnostics of disease de novo but also for guided surgery, PDT dosimetry and therapeutic monitoring. Other uses of fluorescence in PDT (not necessarily from the PDT agents) include the development of technologies that allow tracking of cells during treatment in vivo, studies of sub-cellular localization of molecules for mechanistic studies and photosensitizer tracking for specific targeting. An overview of studies on these aspects from different laboratories will be presented.
Optical technologies, such as reflectance and fluorescence spectroscopy, have shown the potential to provide improved point-of-care detection methods for cervical neoplasia that are sensitive, specific, and cost-effective. Our specific goals are to analyze the diagnostic potential of reflectance and fluorescence spectra, alone and in combination, to discriminate normal and precancerous cervical tissue in vivo and to identify which classification features contain significant diagnostic information. Reflectance spectra are measured at four source-detector separations and fluorescence emission spectra are measured at 16 excitation wavelengths, from 324 sites in 161 patients. These 20 spectral features are permuted in all possible combinations of one, two, and three; and classification algorithms are developed to evaluate the diagnostic performance of each combination. Algorithms based on fluorescence spectra alone yield better diagnostic performance than those based on reflectance spectra alone. The combination of fluorescence and reflectance do not significantly improve diagnostic performance compared to fluorescence alone, except in the case of discriminating high-grade precancers from columnar normal tissue. In general, fluorescence emission spectra at 330- to 360-nm and 460- to 470-nm excitation provide the best diagnostic performance for separating all pairs of tissue categories.
Fluorescence spectroscopy is a promising technology for detection of epithelial precancers and cancers. While age and menopausal status influence measurements in the cervix, other variables do not significantly affect the diagnosis. In this study we examine probe pressure as a variable. A fiber optic probe to measure fluorescence spectra at different calibrated levels of pressure was designed and tested. A pilot study was conducted measuring fluorescence excitation emission matrices in 20 patients at light, medium, and firm pressure. Spectroscopic data were pre-processed and analyzed to compare mean peak intensities as a function of pressure. Further statistical analyses tested for differences in intensities at each excitation/emission wavelength pair. Four providers made measurements from 41 sites; 33 yielded good quality spectroscopic data (22 squamous normal, 7 squamous abnormal, 3 columnar normal, 1 transformation zone) from 17 of 20 patients. At all pressure levels, abnormal tissue showed less fluorescence intensity than normal tissue, and post-menopausal patients showed higher fluorescence intensity than premenopausal patients, consistent with previous analyses. A permutation analysis suggests that pressure does not significantly affect fluorescence intensity or lineshape. While other studies are needed to confirm these findings, this study suggests that fluorescence spectroscopy is a robust technology likely not influenced by fiber optic probe pressure.
Fluorescence spectroscopy has shown promise for the detection of precancerous changes in vivo. The epithelial and stromal layers of tissue have very different optical properties; the albedo is relatively low in the epithelium and approaches one in the stroma. As precancer develops, the optical properties of the epithelium and stroma are altered in markedly different ways: epithelial scattering and fluorescence increase, and stromal scattering and fluorescence decrease. We present an analytical model of the fluorescence spectrum of a two-layer medium such as epithelial tissue. Our hypothesis is that accounting for the two different tissue layers will provide increased diagnostic information when used to analyze tissue fluorescence spectra measured in vivo. The Beer-Lambert law is used to describe light propagation in the epithelial layer, while light propagation in the highly scattering stromal layer is described with diffusion theory. Predictions of the analytical model are compared to results from Monte Carlo simulations of light propagation under a range of optical properties reported for normal and precancerous epithelial tissue. In all cases, the mean square error between the Monte Carlo simulations and the analytical model are within 15%. Finally, model predictions are compared to fluorescence spectra of normal and precancerous cervical tissue measured in vivo; the lineshape of fluorescence agrees well in both cases, and the decrease in fluorescence intensity from normal to precancerous tissue is correctly predicted to within 5%. Future work will explore the use of this model to extract information about changes in epithelial and stromal optical properties from clinical measurements and the diagnostic value of these parameters.
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